The Journal of Neuroscience, July 19, 2006 • 26(29):7555–7564 • 7555

Behavioral/Systems/Cognitive

Involvement of the Anterior Cingulate Cortex in the
Expression of Remote Spatial MemoryCátia M. Teixeira,1,2 Stephen R. Pomedli,1 Hamid R. Maei,1,3 Nohjin Kee,1 and Paul W. Frankland1,3,4
1

Program in Neuroscience and Mental Health, The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8, 2Graduate Program in Areas of Basic andApplied Biology, Universidade do Porto, 4050-345 Porto, Portugal, and 3Department of Physiology and 4Institute of Medical Science, University of Toronto,
Toronto, Ontario, Canada M5S1A8

Although the hippocampus plays a crucial role in the formation of spatial memories, as these memories mature they may become
additionally (or even exclusively) dependent on extrahippocampal structures. However, the identity of these extrahippocampal structures that support remote spatial memory is currently not known. Using a Morris water-maze task, we show that the anterior cingulate
cortex (ACC) plays a key role in the expression of remote spatial memories in mice. To first evaluate whether the ACC is activated after therecall of spatial memory, we examined the expression of the immediate early gene, c-fos, in the ACC. Fos expression was elevated after
expression of a remote (1 month old), but not recent (1 d old), water-maze memory, suggesting that ACC plays an increasingly importantrole as a function of time. Consistent with the gene expression data, targeted pharmacological inactivation of the ACC with the sodium
channel blocker lidocaine blocked expression of remote, but spared recent, spatial memory. In contrast, inactivation of the dorsal
hippocampus disrupted expression of spatial memory, regardless of its age. We further showed that inactivation of the ACC blocked
expression of remote spatial memory in two different mouse strains, after training with either a hidden or visible platform in a constantlocation, and using the AMPA receptor antagonist CNQX. Together, our data provide evidence that circuits supporting spatial memory
are reorganized in a time-dependent manner, and establish that activity in neurons intrinsic to the ACC is critical for processing remotespatial memories.
Key words: hippocampus; spatial learning; Fos; lidocaine; CNQX; memory consolidation

Introduction
Experiments using lesion, electrophysiological, genetic, and neuroimaging approaches have helped to establish that the hippocampus plays an essential role in the formation of spatial memories (O’Keefe and Nadel, 1978; Burgess et al., 2002; Morris et al.,
2003; Nakazawa et al., 2004; Leutgeb et al., 2005). However, as
spatial memories mature they may become additionally (or evenexclusively) dependent on extrahippocampal structures. Perhaps
the best evidence that spatial memories eventually become dependent on extrahippocampal structures comes from neuropsychological studies of two patients: E.P. and K.C. (Teng and
Squire, 1999; Rosenbaum et al., 2000). These patients both suffered substantial damage to the medial temporal lobe that resulted in extensive (or almost complete) lesions of the hippocampus. As in other patients with hippocampal lesions (Smith andMilner, 1981; Cave and Squire, 1991; Maguire et al., 1996), E.P.
and K.C. both have difficulty acquiring new spatial information.

Received March 11, 2006; revised May 28, 2006; accepted May 31, 2006.
This work was supported by grants from the Canadian Institutes of Health Research and the EJLB Foundation.
C.M.T. received support from the Graduate Program in Areas of Basic and Applied Biology and the Portuguese
Foundation for Science and Technology. We thank Sheena Josselyn for comments on previous drafts of this manuscript, and Christy Cole and Adrienne Yeung for technical assistance.
Correspondence should be addressed to Paul W. Frankland, Program in Neuroscience and Mental Health, The
Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8. E-mail: paul.frankland@sickkids.ca.
DOI:10.1523/JNEUROSCI.1068-06.2006
Copyright © 2006 Society for Neuroscience 0270-6474/06/267555-10$15.00/0

However, memory for similar spatial information that was acquired in the remote past is largely spared (Teng and Squire,
1999; Rosenbaum et al., 2000). Although it is a matter of debate
whether these remote, hippocampal-independent spatial memories retain as much detail as those in control subjects (Rosenbaum
et al., 2004; Clark et al., 2005a,b), these studies nonetheless indicate that spatial memories may become, at least partially, supported by extrahippocampal structures over the course of time.
The identity of extrahippocampal regions that may support remote spatial memory is currently not known.
Recently, the medial prefrontal cortex (mPFC) has beenshown to play a key role in the expression of remote memory(Frankland and Bontempi, 2005). The medial prefrontal cortex
consists of several highly interconnected regions, including theanterior cingulate cortex (ACC), and prelimbic and infralimbic
cortices. These regions are reciprocally connected to sensory,
motor, and limbic cortices (Uylings et al., 2003) and they are
therefore ideally situated to integrate and synthesize informationfrom a large number of different sources. Lesions of the mPFC
(including the ACC and prelimbic cortex) disrupted expressionof remote (month old), but not recent (day old), trace eye-blinkconditioning memories (Takehara et al., 2003). Similarly, pharmacological inactivation of the ACC specifically disrupted expression of remote, but not recent, contextual fear (Frankland etal., 2004) and five-arm discrimination (Maviel et al., 2004) memories. To evaluate whether the ACC plays a similar role in spatial

7556 • J. Neurosci., July 19, 2006 • 26(29):7555–7564

memory, we trained mice in the Morris water-maze task (Morris
et al., 1982). Using two complementary approaches, our datasuggest that the ACC plays a specific role in processing remotespatial information. First, we found that the ACC is activatedafter remote, but not recent, probe tests. Second, we found thatinactivation of the ACC blocks expression of remote, but notrecent, spatial memory.

Materials and Methods
Mice. Male offspring from a cross between C57BL/6NTacfBr [C57B6]
and 129Svev [129] mice (Taconic Farms, Germantown, NY) were used in
the majority of these experiments. These mice were bred in our colony at
The Hospital for Sick Children, and were maintained on a 12 h light/darkcycle with access to food and water ad libitum. In one experiment we used
C57B6 mice, purchased from Taconic Farms. All mice were at least 8
weeks of age at the start of experiments, and behavioral procedures wereconducted during the light phase of the cycle. Experiments were conducted blind to the treatment condition of the mouse, and according tolocal animal care protocols.
Water maze and general training procedures. Behavioral testing was
conducted in a circular water-maze tank (120 cm in diameter, 50 cmdeep), located in a dimly lit room. The pool was filled to a depth of 40 cm
with water made opaque by adding white nontoxic paint. Water temperature was maintained at 28 ⫾ 1°C by a heating pad located beneath thepool. A circular escape platform (10 cm diameter) was submerged 0.5 cm
below the water surface and located in the one of the quadrants. For
visible platform experiments, a cylindrical cue (4 cm diameter ⫻ 4 cm)
was added to the platform. The pool was surrounded by curtains, at least
1 m from the perimeter of the pool. The curtains were white, and had
distinct cues painted on them.
Before the commencement of training, mice were individually handled for 2 min each day for 1 week. On each training day, mice received
six training trials (presented in two blocks of three trials; interblockinterval was ⬃1 h; intertrial interval was ⬃15 s). On each trial they wereplaced into the pool, facing the wall, in one of four start locations. The
order of these start locations was pseudorandomly varied throughout
training. The trial was complete once the mouse found the platform or
60 s had elapsed. If the mouse failed to find the platform on a given trial,
the experimenter guided the mouse onto the platform. In these experiments, we used three different water maze training protocols. In thehidden version, the platform was submerged in a fixed location for all
trials. In the visible/fixed version, the platform was marked by a visible
cue and in a fixed location for all trials. In the visible/variable version, theplatform was marked by cue, and its location was pseudorandomly varied on each trial between 4 different positions in the pool. In this version
only, nonpatterned white curtains replaced the curtains containing distalcues to minimize reliance on spatial strategies. After the completion oftraining, spatial memory was assessed in a probe test. In this test, theplatform was removed from the pool, and the mouse was allowed 60 s tosearch for it.
Behavioral data from training and the probe tests were acquired andanalyzed using an automated tracking system (Actimetrics, Wilmette,
IL). Using this software, we were able to record a number of parameters
during training, including escape latency and swim speed. In probe tests,
we quantified performance by measuring the amount of time micesearched the target zone (23 cm in radius, centered on the location of theplatform during training) versus the average of three other equivalentzones in other areas of the pool (Moser et al., 1993; Moser and Moser,
1998; de Hoz et al., 2004). These zones each represent ⬃15% of the total
pool surface. Two additional analyses were performed. First, to evaluate
the precision of search patterns (Moser et al., 1993), we quantified exploration in three concentric circles (radii: 16 cm, 23 cm, and 28 cm) centered on the platform location. The regions represented by the inner,middle, and outer circles are of equal area (with each representing ⬃7%of the total pool surface). Second, to evaluate thigmotaxic behavior, we
calculated the amount of time mice spent in the peripheral region of thepool (the area within 6 cm of the wall) (Martin et al., 2005). This peripheral region represents ⬃19% of the total pool surface.

Teixeira et al. • The Anterior Cingulate Cortex and Spatial Memory

Fos immunocytochemistry. In experiments examining Fos expression
in the ACC, mice were perfused transcardially with 0.1 M PBS and then
4% paraformaldehyde (PFA) 90 min after the completion of behavioral
testing. Brains were removed, fixed overnight in PFA, and then transferred to 30% sucrose solution and stored at 4°C. Fifty-micrometer coronal sections were cut and free-floating sections were prepared for immunocytochemistry using anti-Fos primary rabbit polyclonal antibody (1:20000, Rabbit anti-Fos polyclonal IgG; Oncogene Research Products,
Darmstadt, Germany) and a biotinylated goat anti-rabbit antibody (1:2000, Biotin-SP-conjugated affiniPure Goat anti-rabbit IgG; Jackson ImmunoResearch, West Grove, PA) was used as a secondary. Staining was
revealed using the avidin-biotin peroxidase method (Vectastain ABC kit;
Vector Laboratories, Burlingame, CA) coupled to diaminobenzidine as
chromogen. Quantitative analysis of Fos-positive nuclei was performed
using the Image J processing system. Structures were anatomically defined according to a mouse brain atlas (Paxinos and Franklin, 2000).
Because we found no evidence for lateralization of Fos expression in the
ACC in our initial analyses (left, 7.72 ⫾ 0.94; right, 7.14 ⫾ 1.11; F(1,5) ⫽1.35; p ⬎ 0.05), immunoreactive neurons were counted unilaterally
along the entire anterior-posterior extent of this region. In addition, Fos
expression was quantified in subregions of the hippocampus (CA1, CA3,and dentate gyrus).
Surgery and pharmacological inactivation. Under chloral hydrate anesthesia and using standard stereotaxic procedures, stainless-steel guidecannulas (22 gauge) were implanted into the ACC (⫹0.8 mm, 0 mm,⫺1.0 mm) or the dorsal hippocampus (dHPC) (⫺2.0 mm, ⫾2.0 mm,⫺2.0 mm). Mice were allowed to recover for at least 1 week after surgery.
Ten minutes before probe tests, mice received 0.5 ␮l infusions of either
drug or vehicle (PBS) at a rate of 0.2 ␮l/min. In the majority of experiments, we used lidocaine (4%; Sigma, St. Louis, MO). In one experiment,
we used 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 3 mM; Tocris,
Ellisville, MO). The injection cannula (28 gauge) was left in place for 2
min after the infusion and the mice were returned to their home cages
before testing.
Anatomical specificity is a critical issue when using pharmacological
techniques to inactivate brain regions, and we used two approaches toevaluate both location and extent of infusion. First, in all mice cannula
placement was verified histologically. At the completion of the experiments, mice were overdosed with chloral hydrate and perfused transcardially with saline, followed by 4% PFA solution to fix the brain tissue.
Brains were removed, postfixed in 4% PFA solution, and cut into 50 ␮m
coronal sections on a cryostat. Sections were mounted on slides, stainedwith neutral red, and cannula placements were examined under a lightmicroscope. Second, we examined Fos expression after water-maze testing in a subset of mice treated with CNQX. Using the same quantification
methods described above, the extent of inactivation in CNQX-treatedmice was estimated by defining a region where Fos expression was reduced by 50% or more relative to PBS-treated mice. Using this method,
we found that Fos expression was reduced in a roughly circular region
1.0 –1.5 mm in diameter, below the tip of the injector (see Fig. 6e–f ). This
indicates that infusions were largely restricted to the ACC, with limited
diffusion to neighboring structures.
Experimental procedures. In the first experiment, we examined whether
spatial memory testing induced Fos expression in the ACC. In this experiment, separate groups of mice were trained in either the visible/fixed or
the visible/variable version of the water maze for 5 d. Then either 1 d(recent test: visible/fixed, n ⫽ 8; visible/variable, n ⫽ 8) or 30 d (remote
test: visible/fixed, n ⫽ 8; visible/variable, n ⫽ 8) later they were given aprobe test. In the second experiment, examining whether inactivation of
the ACC or dHPC disrupted spatial memory expression, mice weretrained in the hidden version of the water maze for 3 d. Spatial memory
was then probed 1 d (PBS, n ⫽ 13; ACC, n ⫽ 10; dHPC, n ⫽ 9) or 30 d
(PBS, n ⫽ 12; ACC, n ⫽ 9; dHPC, n ⫽ 10) later. Roughly half of the micein the PBS group received infusions into the ACC, whereas the others
received infusions into the dHPC. There were no differences between
these two groups, and so they were combined. In the third experiment,
C57B6 mice were trained for 7 d in the hidden version of the water maze.
Spatial memory was then tested either 1 d (PBS, n ⫽ 19; ACC, n ⫽ 9;
dHPC, n ⫽ 7) or 30 d (PBS, n ⫽ 23; ACC, n ⫽ 8; dHPC, n ⫽ 9) later.

Teixeira et al. • The Anterior Cingulate Cortex and Spatial Memory

J. Neurosci., July 19, 2006 • 26(29):7555–7564 • 7557

Figure 1. The ACC is activated after expression of remote spatial memory. a, Mice were trained to locate a visible platform in a fixed location in the water maze. Over training, mice assigned to
the recent or remote testing groups learned to find the escape platform with decreasing latencies. b, Spatial memory was evaluated in a probe test either 1 d (recent group) or 30 d (remote group)
after training. Both groups of mice spent more time searching the target zone (T) compared with other (O) zones in this test. c, Fos expression was elevated in the ACC after the remote compared withthe recent probe test (number of Fos-positive nuclei per 10,000 ␮m 2). Representative sections show Fos expression in the ACC after a recent (d) and remote (e) probe test. Scale bars: 100 ␮m. f,
In the control condition, mice were trained to locate a visible platform, whose position varied from trial to trial. Over training, mice assigned to the recent or remote testing groups learned to find thevisible platform with decreasing latencies. g, Spatial memory was evaluated in a probe test either 1 d (recent group) or 30 d (remote group) after training. Neither group searched selectively in thetarget zone compared with other zones in these tests. h, Levels of Fos expression in the ACC were equivalent after the recent and remote probe tests. Representative photomicrographs show Fos
expression in the ACC after a recent (i) and remote (j) probe test. Scale bar, 100 ␮m. k, Detailed analysis of Fos expression along rostral-caudal extent of the ACC in mice trained in the visible/fixed(left) and visible/variable (right) versions of the water maze. Distances relative to bregma are shown in millimeters. l, Detailed analysis of Fos expression in different layers in the ACC in mice trainedin the visible/fixed (left) and visible/variable (right) versions of the water maze. m, Comparison of training performance in the visible/fixed (V/F) and visible/variable (V/V) versions of the water maze.
During training there were no differences in the mean latency to find the escape platform (left) or the average number of reinforced trials (i.e., when mice successfully found the platform) (right).
Error bars indicate SE.
Again, roughly half of the mice in the PBS group received infusions into
the ACC, whereas the others received infusions into the dHPC. There
were no differences between these two groups, and so they were combined. In the fourth experiment, mice were trained in the visible/fixedversion of the water maze for 5 d, and then their spatial memory wastested 30 d later after infusions of either PBS (n ⫽ 9) or lidocaine (n ⫽ 11)
into the ACC. In the final experiment, mice were trained in the hidden
version for 3 d. Their spatial memory was assessed in a probe test 30 dlater. Before this test, they received infusions of PBS (n ⫽ 7) or CNQX(n ⫽ 8) into the ACC. Four days later, they were given a second probe
test, this time drug-free. After a further 20 d, they received two more
drug-free probe tests: one probe test was conducted with the visual distalcues present, another probe test with the visual distal cues obscured byadding a black curtain around the perimeter of the pool. The order ofthese tests counterbalanced.

Results
The ACC is preferentially activated after expression of remotespatial memory
The expression of the immediate early gene, c-fos, is correlatedwith levels of neuronal activity and can be used to track changes
in the organization of memories at different times after learning(Kaczmarek and Robertson, 2002). Therefore, we first used Fos

immunocytochemistry to evaluate whether the ACC is preferentially activated after expression of remote spatial memory. Mice
were initially trained to find a visible platform in a fixed locationin the pool, and then memory was assessed in separate groups ofmice in probe tests either 1 d (recent test) or 30 d (remote test)
later. Over the 5 d of training, mice required progressively lesstime to escape to the platform (F(4,56) ⫽ 69.44; p ⬍ 0.001) (Fig.
1a), and there were no differences in escape latencies between
mice assigned to the recent and remote groups [no significant
effect of group (F(1,14) ⬍ 1) or group by training day interaction(F(4,56) ⬍ 1)].
This training schedule produced a stable and long-lasting spatial memory; mice showed a strong spatial bias for the area of thepool where the platform was located during training in the recent
and remote probe tests. We quantified this spatial bias by comparing how much time mice spent searching the target zone (a 23
cm radius circular zone centered on the former platform location) versus the average time they spent searching equivalentzones in the other three quadrants (Moser et al., 1993; Moser andMoser, 1998; de Hoz et al., 2004). ANOVA with group (recent vsremote) as a between-subjects variable and zone (target vs others)

7558 • J. Neurosci., July 19, 2006 • 26(29):7555–7564

Teixeira et al. • The Anterior Cingulate Cortex and Spatial Memory

as a within-subjects variable revealed a significant main effect of zone (F(1,14) ⫽21.96; p ⬍ 0.001), indicating that micespent more time exploring the target zone
compared with other zones. There was no
significant effect of group (F(1,14) ⬍ 1) orgroup by zone interaction (F(1,14) ⬍ 1),
indicating that mice performed similarlyin the recent and remote tests (Fig. 1b).
Although performance was similar in the Figure 2. Fos expression in the hippocampus after recent and remote probe tests. Fos expression after recent and remote probe(V/F; black bars) or visible/variable (V/V; white bars)
recent and remote probe tests, we found tests in CA1 (a), CA3 (b), and DG (c) after training in either the visible/fixedversions of the water maze (number of Fos-positive nuclei per 10,000 ␮m 2). The hatched bars show Fos levels in the same regionsthat Fos expression in the ACC was signif- for home cage (HC) control mice. Error bars indicate SE.
icantly higher after the remote, comparedwith the recent, probe test (F(1,13) ⫽ 27.39,
Fos expression in the hippocampus after recent and remotep ⬍ 0.001) (Fig. 1c– e). This increase was evident throughout theprobe tests
rostral-caudal extent of the ACC, and was not limited to a particWe also examined Fos expression in the hippocampus after theular layer, or subset of layers, within the ACC (Fig. 1k–l ).
recent and remote probe tests for mice trained in the visible/fixed
These results suggest that the ACC is preferentially involved in
and visible/variable versions of the water maze (Fig. 2). In each of
the processing of remote (compared with recent) spatial inforthe hippocampal subregions, Fos expression was similar in themation. However, mice in the remote test group also had considrecent and remote probe tests, regardless of the type of training:erably less recent experience of being handled, swimming in the
CA1 [no main effects of training (F(1,27) ⫽ 2.26; p ⬎ 0.05), retenwater maze, and so on. Therefore, it is possible that increased Fostion (F(1,27) ⫽ 2.10; p ⬎ 0.05) or training by retention interaction
expression might be confounded by nonspecific increases in(F(1,27) ⬍ 1)], CA3 [no main effects of training (F(1,27) ⬍ 1),
arousal associated with the remote probe test. To control for this,retention (F(1,27) ⫽ 1.29; p ⬎ 0.05) or training by retention interaction (F(1,27) ⬍ 1)], and dentate gyrus (DG) [no main effects of
we trained a second group of mice in a visible/variable version on
training (F(1,27) ⫽ 3.80; p ⬎ 0.05), retention (F(1,27) ⫽ 2.17; p ⬎the water maze. As in the previous experiment, the platform was0.05) or training by retention interaction (F(1,27) ⫽ 1.56; p ⬎marked by a visible cue. However, because (1) the position of the0.05)]. In every case, levels of Fos expression in these tested mice
platform was pseudorandomly varied between four possible lowere significantly greater than those in home-cage controls (n ⫽cations on each trial, and (2) the curtains containing distal cues
5; t tests, all p values ⬍ 0.05), suggesting that performance (e.g.,
were replaced with featureless white curtains, this version of thenavigation), as well as mnemonic, aspects of water-maze testingwater maze discourages the development of spatial strategies.
contribute to activation of the hippocampus.
When trained in this way, mice required progressively less time toescape to the platform (F(4,56) ⫽ 34.62; p ⬍ 0.001) (Fig. 1f ), andthere were no differences in escape latencies between mice assigned to the recent and remote groups [no significant group(F(1,14) ⬍ 1) or group by training day interaction (F(4,56) ⬍ 1)].
Consistent with the idea that training in the visible/variable version of the water maze discourages the development of a spatialbias, mice spent equivalent time searching in the target zone compared with other zones [no significant group (F(1,14) ⬍ 1), zone
(F(1,14) ⫽ 1.34; p ⫽ 0.27) or group by zone interaction (F(1,14) ⬍1)] in the probe tests either 1 d or 30 d after training (Fig. 1g).
Most importantly, after this type of training, levels of Fos expression in the ACC were equivalent after the recent and remoteprobe tests (F(1,13) ⫽ 1.11; p ⫽ 0.31) (Fig. 1h–j). These results,
therefore, indicate that the increased Fos expression after remotememory testing in the first experiment is most likely related to theremoteness of the spatial memory, and not to differences inarousal at the recent and remote time points.
It is worth noting that this visible/variable version of thewater-maze controls for both swimming experience and reinforcement during the training period. Both the amount of time
taken to swim to the platform (visible/fixed, 11.73 ⫾ 1.04; visible/
variable, 12.61 ⫾ 1.20; F(1,28) ⬍ 1), as well as the frequency ofreinforcement (visible/fixed, 28.63 ⫾ 0.54; visible/variable,
28.38 ⫾ 0.63; F(1,28) ⬍ 1) were equivalent during training in thetwo versions (Fig. 1m). Other types of control conditions havebeen used, including swimming controls (Gusev et al., 2005).
However, whereas swimming controls equate swimming experience, reinforcement differs between groups and this may lead todifferences in motivation and/or stress levels.

Inactivating the ACC disrupts expression of remotespatial memory
We next tested whether pharmacological inactivation of the ACC
or dHPC disrupts expression of spatial water-maze memories. In
this experiment, separate groups of mice were trained over 3 d tolocate a hidden platform. During training, escape latencies declined over days (data not shown; F(2,114) ⫽ 166.31; p ⬍ 0.001).
After the completion of training, spatial memory was assessed ina probe test either 1 d (recent test) or 30 d (remote test) later.
Mice treated with PBS before the probe test exhibited strong
memory at both retention delays (Fig. 3a). ANOVA with group(recent vs remote) as a between-subjects variable and zone (target
vs others) as a within-subjects variable was performed on theprobe test data for the PBS-treated mice. This analysis revealed asignificant main effect of zone (F(1,23) ⫽ 40.62; p ⬍ 0.001), indicating that mice searched selectively in the target zone that previously contained the escape platform. There was no significant
group effect (F(1,23) ⫽ 1.36; p ⫽ 0.26) or group by zone (F(1,23) ⬍1) interaction, indicating that PBS-treated mice performed similarly in the recent and remote tests. In contrast, lidocaine infusions into the ACC disrupted performance in the remote, but notrecent, probe test (Fig. 3b). ANOVA revealed significant main
effects of both group (F(1,17) ⫽ 8.69; p ⬍ 0.01) and zone (F(1,17) ⫽15.65; p ⬍ 0.01) and, most importantly, a significant group byzone interaction (F(1,17) ⫽ 9.04; p ⬍ 0.01), indicating that micespent more time searching the target zone (vs the other zones) in
the recent, but not remote, probe test. Post hoc analyses (Neuman–Keuls; p values ⬍ 0.05) confirmed that lidocaine infusions
into the ACC only disrupted performance in the 30 d, and not theTeixeira et al. • The Anterior Cingulate Cortex and Spatial Memory

J. Neurosci., July 19, 2006 • 26(29):7555–7564 • 7559

revealed mice in the PBS groups spent significantly more time in the target zone
than those in either the ACC or dHPCgroups.
We next explored the nature of the deficits produced by these targeted inactivations in more detail. In initial training trials, naive mice tend to spend the majorityof their time searching the periphery of thepool (Wolfer et al., 1998). This thigmotaxic behavior tends to decline rapidly
over training (Fig. 4), suggesting that overtime mice learn more effective searchstrategies (Martin et al., 2005). In the current study, we observed a similar decline
in thigmotaxic behavior in mice in the
PBS-treated group (Fig. 4a): the amountof time spent searching in the peripheral
region of the pool (within 6 cm of the wall)
declined dramatically from the first training trial (recent group, 57.27 ⫾ 2.63%; remote group, 62.27 ⫾ 2.74%) to the probe
test (recent group, 4.11 ⫾ 3.11%; remotegroup, 9.70 ⫾ 3.23%) (main effect oftraining: F(1,23) ⫽ 264.34; p ⬍ 0.001).
A similar decline occurred in mice in
the ACC/lidocaine group (main effect oftraining: F(1,17) ⫽ 114.33; p ⬍ 0.001) (Fig.
4b), suggesting that inactivation of the
ACC does not interfere with memory forprocedural aspects of the task. The decline
was less pronounced in the remote memory group (training, 53.81 ⫾ 5.25%; probe
test, 18.79 ⫾ 3.82%). However, the time
Figure 3. Effects of lidocaine inactivation of ACC and dHPC on recent and remote spatial memory. Spatial memory was evalu- spent exploring this outer region in theated in a probe test either 1 d (recent group) or 30 d (remote group) after training in the hidden version of the water maze. Ten probe is what would be predicted by a ranminutes before testing, mice received infusions of either PBS (a) or lidocaine into the ACC (b) or dHPC (c). Left, Percentage of time dom search pattern because this outer respent searching the target zone (T) compared with other (O) zones in probe tests. Right, Density plot for grouped data showing gion represents ⬃19% of the total surface
where mice concentrated their searches in the recent and remote probe tests, respectively. The color scale represents the numberof the pool (t(8) ⫽ ⫺0.04; p ⫽ 0.97).
of visits per animal per 5 ⫻ 5 cm area. Error bars indicate SE.
In the dHPC/lidocaine group, thigmotaxic behavior also declined from the first
1 d, probe test. Inactivation of the dHPC disrupted performancetraining trial to the probe test (F(1,17) ⫽ 51.70; p ⬍ 0.001) (Fig.
4c). Again, these mice tended to spend more time exploring thein both the recent and remote memory tests (Fig. 3c). ANOVA
periphery of the pool in the probe tests compared with PBSrevealed no main effects of either group (F(1,17) ⬍ 1) or zone
(F(1,17) ⫽ 2.77; p ⫽ 0.11). Although there was a significant grouptreated mice (recent, 32.26 ⫾ 6.56%; remote, 24.85 ⫾ 6.22%).
by zone interaction (F(1,17) ⫽ 5.41; p ⬍ 0.05), post hoc analyses
Nonetheless, these values do not differ significantly from whatindicated that mice did not search significantly more in the target
would be expected for a random search pattern in either caseversus other zones in either the recent or remote probe tests(recent: t(8) ⫽ 2.12, p ⫽ 0.07; remote: t(9) ⫽ 0.90, p ⫽ 0.39).
Together, these analyses suggest that these targeted inactivations(Neuman–Keuls; p values ⬍ 0.05). Finally, to examine the relahave selective effects on spatial memory and spare memory fortive effects of inactivation of the ACC and dHPC on the expresprocedural aspects of the task (i.e., mice remember to avoid thesion of recent and remote spatial memory, we conducted
walls).
ANOVA with infusion (PBS, ACC, and dHPC) as a betweenIn some tasks, inactivating or lesioning the dHPC produces
subjects variable and zone (target vs others) as a within-subjects
temporally graded effects on memory, with recent memories afvariable. For the recent probe tests, these analyses revealed signiffected more profoundly than remote memories (Squire and Alicant main effects of infusion (F(2,29) ⫽ 11.66; p ⬍ 0.001) andzone (F(1,29) ⫽ 40.02; p ⬍ 0.001), as well as a significant infusion
varez, 1995; Squire et al., 2001; Frankland and Bontempi, 2005).
by zone interaction (F(2,29) ⫽ 3.92; p ⬍ 0.05). Post hoc analyses
Our analysis suggested inactivation of the dHPC has equivalentrevealed mice in the PBS and ACC groups spent significantly
effects on performance in both the recent and remote probe tests.
more time in the target zone than those in the dHPC group. For
However, it is possible that inactivation of the dHPC results in
the remote probe tests, these analyses revealed significant mainless precise, but nonetheless partially spared, spatial preference in
effects of infusion (F(2,28) ⫽ 8.20; p ⬍ 0.01) and zone (F(1,28) ⫽the remote test (Winocur et al., 2005). To evaluate whether there
11.38; p ⬍ 0.01), as well as a significant infusion by zone interacis any evidence of a graded effect, we examined the precision oftion (F(2,28) ⫽ 10.01; p ⬍ 0.001). In this case, post hoc analyses
search patterns in the recent and remote probe tests (Moser et al.,

7560 • J. Neurosci., July 19, 2006 • 26(29):7555–7564

Teixeira et al. • The Anterior Cingulate Cortex and Spatial Memory

Figure 4. Detailed analysis of performance in recent and remote probe tests. Detailed analysis is shown for mice receiving infusions of PBS (a) or lidocaine into the ACC (b) or dHPC (c), respectively.
Swim paths from the first training trial and probe tests are shown for representative mice in the recent and remote test groups, respectively. The position of the platform during training is marked
in the lower right quadrant. In addition, a concentric circle indicates the peripheral region of the pool used to quantify thigmotaxic behavior. The graph on the left shows the amount of time spentin the periphery of the pool during the first training trial versus probe test for mice in the recent and remote test groups. The graph on the right shows the precision of search pattern indicated by timespent searching equal area regions marked by inner, middle, and outer circles centered on the position of the platform during training (for details, see Results). Error bars indicate SE.

1993). To do this we quantified exploration in three concentric
circles (radii: 16 cm, 23 cm and 28 cm) centered on the platform
location. The areas represented by the inner, middle, and outerzones are of equal area, and more precise searches should produce more pronounced searching in the inner versus middle versus outer zones, respectively. PBS-treated mice had precise searchpatterns, spending more time exploring the inner versus middle
versus outer zones, respectively, in the probe tests (significant
main effect of zone: F(2,46) ⫽ 42.73; p ⬍ 0.001) (Fig. 4a). There
was no significant main effect of group (F(1,23) ⬍ 1) nor group byzone interaction (F(2,46) ⫽ 1.45; p ⫽ 0.25), suggesting thatsearches were equivalently precise at both time points. Mice receiving lidocaine infusions into the ACC only showed this precise
searching pattern in the recent probe test (Fig. 4b). ANOVA revealed significant main effects of both group (F(1,17) ⫽ 9.81; p ⬍0.01) and zone (F(2,34) ⫽ 20.76; p ⬍ 0.001) and, most importantly, a significant group by zone interaction (F(2,34) ⫽ 4.16; p ⬍0.05), confirming that lidocaine infusions into the ACC only disrupted performance in the remote, and not the recent, probe test.
Analysis of probe test data in mice receiving lidocaine infusions into the dHPC provided no evidence for a graded effect on
recent versus remote memory. Rather, mice spent equivalentamounts of time in each of the three zones in the probe tests(zone: F(2,34) ⫽ 1.16; p ⫽ 0.32) (Fig. 4c). This was the case in both
the recent and the remote probe test [no significant group byzone interaction (F(2,46) ⬍ 1)], suggesting that lidocaine inactivation of dHPC did not differentially affect the precision of recent
versus remote spatial memory.
Inactivation of the ACC disrupts expression of remote spatial
memory in C57B6 mice
Performance in learning tasks such as the Morris water maze isinfluenced by genetic background (Crawley et al., 1997). There-

fore, to explore the generality of our findings, we also examinedthe effects of these same manipulations in a different mousestrain (inbred C57B6 mice). In this independent series of studies
we found that lidocaine-induced inactivation of the ACC anddHPC produced exactly the same pattern of results (supplemental Fig. 1, available at www.jneurosci.org as supplemental material), suggesting that the roles of these regions in the expression of
spatial memory are conserved across different genetic
backgrounds.
Inactivation of the ACC disrupts expression of remote spatial
memory after training with fixed, visible platform
In our initial immunocytochemical studies, mice were trained tofind a fixed platform that was marked by a visible cue. So far, weonly examined the effects of ACC inactivation on spatial memory
expression after training in a hidden version of the water maze.
Therefore, we next tested whether ACC inactivation impairs remote memory expression after training with a fixed, visible platform. During training, mice required progressively less time toescape to the platform (F(4,72) ⫽ 74.69; p ⬍ 0.001), and there were
no differences in escape latencies between groups [no significant
main effect of group (F(1,18) ⬍ 1) or group by drug interaction(F(4,72) ⬍ 1)]. Inactivation of the ACC with lidocaine disrupted
performance in the remote memory probe test 30 d after training(Fig. 5). Whereas PBS-treated mice spent significantly more time
exploring the target compared with other zones, this spatial bias
was less pronounced in the lidocaine treated mice (significant
group by zone interaction: F(1,18) ⫽ 5.64; p ⬍ 0.05). Post hoc
analyses showed that only the PBS-treated mice spent more time
exploring the target zone compared with other zones ( p ⬍ 0.05).
These data indicate that inactivation of the ACC with lidocaine
disrupts expression of remote spatial memory, regardless ofwhether the platform was visible or hidden during training. Im-

Teixeira et al. • The Anterior Cingulate Cortex and Spatial Memory

J. Neurosci., July 19, 2006 • 26(29):7555–7564 • 7561

mediately after the probe test, PBS- and lidocaine-treated mice
were given a second test, where the escape platform was placedback into the pool and marked by a visible cue. In this test, both
PBS- and lidocaine-treated mice found the platform quickly, andthere was no difference in their escape latencies (t(17) ⫽ 1.15; p ⫽0.27; data not shown), indicating that lidocaine infusions did notinterfere with sensory and motivational requirements of this task.

During training, mice learned to find the platform (F(2,26) ⫽64.03; p ⬍ 0.001), and there were no differences in escape latencies between mice assigned to the PBS and CNQX drug groups[no significant group (F(1,13) ⫽ 2.08; p ⫽ 0.17) or group by training day interaction (F(2,26) ⫽ 1.66; p ⫽ 0.21)]. CNQX infusion
before the probe test blocked expression of remote spatial memory (significant group by zone interaction: F(1,13) ⫽ 5.00; p ⬍0.05) (Fig. 6a), indicating that CNQX produces similar effects tolidocaine when infused into the ACC. Post hoc analyses confirmed that whereas PBS-treated mice searched selectively,
CNQX-treated mice spent equivalent time searching the target
versus other zones ( p ⬍ 0.05). These results suggest that activityof intrinsic excitatory neurons in the ACC is critical for the expression of remote spatial memory.
CNQX infusion suppresses neural activity for ⬃60 min (Dayet al., 2003) and, therefore, these infusions should only transiently disrupt expression of spatial memory. To test whether theeffects of the CNQX infusions were reversible, mice were tested
drug-free 4 d later. In this test, both groups of mice (those previously treated with CNQX and those previously treated with PBS)
searched selectively in the target area of the pool for the platform[significant main effect of zone: F(1,13) ⫽ 26.31, p ⬍ 0.001; nosignificant group (F(1,13) ⫽ 2.55; p ⫽ 0.13), or group by zone
interaction (F(1,13) ⫽ 1.21; p ⫽ 0.29)] (Fig. 6b), indicating that theeffects of CNQX were reversible, and likely did not produce any
permanent damage in the ACC.

Inactivation of the ACC with CNQX reversibly disrupts
expression of remote spatial memory
Lidocaine is a sodium channel blocker and therefore suppresses
neuronal activity of both excitatory and inhibitory neurons in the
ACC, as well as fibers of passage (Sandkuhler et al., 1987). To
evaluate whether excitatory transmission is necessary for expression of remote spatial memories, we used the AMPA receptor
antagonist, CNQX (Day et al., 2003). Mice were trained for 3 d in
the hidden version of the water maze, and then tested 30 d later.

Performance in remote probe tests depends on distal
visual cues
Targeted search strategies in the water maze depend on the formation of an accurate, allocentric representation of the distalcues surrounding the maze (O’Keefe and Nadel, 1978; Eichenbaum et al., 1990). To test whether remote spatial memory depended on these distal cues in our water maze, we performed two
additional probe tests on the mice above with these distal cues
either (1) present or (2) obscured by a black curtain. All mice

Figure 5. Effects of inactivating the ACC on remote spatial memory after training with fixed,
visible platform. Spatial memory was evaluated in a probe test 30 d after training. The percentage of time spent searching the target zone (T) compared with other (O) zones in the probe
test is shown for mice that received infusions of either PBS or lidocaine into the ACC. Error
bars indicate SE.

Figure 6. Effects of CNQX inactivation of ACC on remote spatial memory. In this experiment, mice were trained in the hidden version of the water maze, and their spatial preference evaluated ina series of probe tests. Thirty days after training, mice were given a remote probe test after intra-ACC infusions of either PBS or CNQX (a). These mice were subsequently retested 4 d later, drug-free(b). Finally, these same mice were given two more probe tests: one in which the distal visual cues present (c), the other with these cues obscured by a black curtain (d). For each of these probe tests,
the percentage of time spent searching the target zone (T) compared with other (O) zones is shown on the left. Additionally, density plots for grouped data showing where searches wereconcentrated in the PBS (upper) and CNQX (lower) groups are shown on the right. The color scale represents the number of visits per animal per 5 ⫻ 5 cm area. Representative photomicrographsof Fos expression in the ACC after water-maze testing shown for mice pretreated with either PBS (e) or CNQX (f ). Scale bars, 100 ␮m. Error bars indicate SE.

7562 • J. Neurosci., July 19, 2006 • 26(29):7555–7564

were tested drug free. As expected, with the distal cues present,
both groups of mice concentrated their search in the target zone
compared with other zones (significant main effect of zone:
F(1,13) ⫽ 22.80; p ⬍ 0.001) (Fig. 6c). However, when these cues
were obscured, neither group searched selectively (zone: F(1,13) ⫽1.46; p ⫽ 0.25) (Fig. 6d). The order of the two tests (cues present
vs cues obscured) was counterbalanced, and so the lack of spatial
bias in the probe test with the cues removed cannot be attributed
to extinction. Rather, these results indicate that spatial performance in these probe tests depends on an allocentric representation of the extramaze distal cues.

Discussion
Our results establish a role for the ACC in the expression of
remote spatial memory. Using a water-maze task, we found that
increased activation of the ACC correlated with expression of
remote (but not recent) spatial memory. Conversely, we foundthat inactivating the ACC disrupted performance only in tests ofremote spatial memory. This disruption of remote memory wasobserved in two different mouse strains after training with eithera hidden or visible platform in a constant location, and using
either lidocaine or the AMPA receptor antagonist CNQX. Together, these results establish that activity in neurons intrinsic to
the ACC is critical for processing remote spatial memories.
The hippocampus and remote spatial memory
Previous studies have examined the impact of post-training hippocampal lesions on water-maze memory (Bolhuis et al., 1994;
Mumby et al., 1999; Sutherland et al., 2001; Clark et al., 2005a,b;Martin et al., 2005). In each of these studies, hippocampal lesions
disrupted spatial memories, regardless of the interval between
training and surgery. This was the case even after extensive pretraining in the water maze (Clark et al., 2005b), partial lesions ofhippocampus (Clark et al., 2005a; Martin et al., 2005), when extended delays between training and surgery were used (Clark etal., 2005a), when water mazes with reduced spatial complexity
were used (Clark et al., 2005a), or when reminders to aid retrieval
were used (Martin et al., 2005). Similarly, we found no evidence
for a graded effect on recent versus remote spatial memory after
inactivation of the dorsal hippocampus, consistent with a recent
study using similar inactivation approaches in rats (Broadbent etal., 2006).
This temporally ungraded pattern is distinct from the timedependent role of the hippocampus in some other tasks that also
involve processing spatial information [e.g., contextual fear conditioning (Kim and Fanselow, 1992; Anagnostaras et al., 1999);
5-arm discrimination (Maviel et al., 2004)]. In particular, using
similar approaches, we previously showed that the hippocampus
is activated after recent, but not remote, contextual fear memory
testing (Frankland et al., 2004), suggesting that the hippocampus
plays only a temporary role in the expression of contextual fear
memories. In contrast, our gene expression and inactivation
studies suggest that the hippocampus is continuously needed forthe expression of a water-maze memory. There are perhaps two
key differences between the water maze and contextual fear conditioning that may account for this discrepancy.
First, the hippocampus may be important for the expressionof spatial memories that require animals to navigate throughspace, as in the water maze. In such tasks, hippocampal activity
may be required to continuously integrate both idiothetic (selfmotion cues) and allothetic (e.g., distal visual cues) information
to successfully navigate to the escape platform (for discussion, see
Clark et al., 2005a; Martin et al., 2005). Consistent with this, we

Teixeira et al. • The Anterior Cingulate Cortex and Spatial Memory

found robust hippocampal Fos expression in both the visible/
fixed and visible/variable groups, suggesting that navigational demands of the water maze alone significantly engage the hippocampus. Presumably, abolition of this activity interferes withthe expression of remote (as well as recent) water maze memories, in part, by affecting the animal’s ability to integrate idiothetic
and allothetic information. Second, the hippocampus may always be necessary for rich, detailed spatial representation of environments (Rosenbaum et al., 2000; Winocur et al., 2005;
Moscovitch et al., 2006), and such precise representations may beessential for the continued expression of a water-maze memory.
This contrasts with contextual fear conditioning where mice tend
to show generalized freezing at remote time-points (Balogh et al.,2002), suggesting that as these memories age they become lessprecise. The important distinction here is that although precision(and presumably hippocampal involvement) may not be necessary for the continued expression of a contextual fear memory, it
is perhaps essential for the expression of a water-maze memory inwhich mice must remember a specific location in an otherwise
relatively featureless environment. The application of pharmacological and/or genetic approaches that can temporally limit disruption to either the consolidation period or to testing will help
to discriminate between these performance (navigation) andmnemonic (precision) accounts of water-maze deficits after hippocampal inactivation (Riedel et al., 1999; Wang et al., 2003; Cuiet al., 2004; Micheau et al., 2004).
The ACC and remote spatial memory
Although we found that the hippocampus is always required forthe expression of water-maze spatial memories, our key finding isthat as these memories mature they become additionally dependent on the ACC. The effects of ACC inactivation on memory
expression were selective, not only in terms of the remoteness ofthe memory to be retrieved, but also in terms of the type ofmemory affected. Successful performance in a probe test depends
on a number of potentially dissociable memory systems(Micheau et al., 2004); an animal must remember both where tosearch and also how to search. More detailed analysis of probe
test performance showed that ACC inactivation appeared tospare more procedural (how to) aspects of water maze memory.
For example, lidocaine-treated mice did not behave like naive
mice in the remote probe test. Rather, these mice still spent substantially less time in the periphery of the pool than they did at the
beginning of training, indicating that memory for certain strategies (such as avoiding the walls of the pool) was not affected. In
addition, lidocaine inactivation of the ACC did not impair theability of mice to swim to a marked platform, indicating thatthese infusions did not interfere with sensory and motivational
requirements of this task.
These data begin to identify a set of regions that play essential
roles in the continued expression of a spatial memory, and suggest that circuits supporting expression of these spatial memories
are reorganized in a time-dependent manner. We found thateliminating activity in the dHPC or the ACC completely disrupted expression of remote spatial memory. Also likely to play
an essential role is the entorhinal cortex (Remondes and Schuman, 2004; Steffenach et al., 2005), a region that sits at the interface between the hippocampus and neocortex. For example, lesions of the dorsolateral band of the entorhinal cortex disrupt
expression of a previously acquired spatial memory (Steffenach etal., 2005). These regions participate in what is very likely to be amuch broader forebrain network that contributes to different
aspects of spatial cognition. For example, this network may in-Teixeira et al. • The Anterior Cingulate Cortex and Spatial Memory

clude the retrosplenial and parietal cortices, regions that play
important roles in representing spatial information in allocentricand egocentric frameworks, respectively (Moscovitch et al.,2005). In addition, this network may include the infralimbic andprelimbic cortices because neural activity in these regions correlates with motivational aspects of performance (e.g., representation of goal locations) (Hok et al., 2005). However, whereas inactivation of the ACC or dHPC completely abolished remotespatial memories, lesions of the retrosplenial, parietal, infralimbic, and prelimbic regions typically only have mild effects on
spatial behavior (Warburton et al., 1998; Aggleton et al., 2000;
Hoh et al., 2003). This suggests that the ACC and the hippocampus may play more essential roles in distributed forebrain networks supporting remote spatial memory.
The role identified here for the ACC in remote spatial memory
parallels its role in remote memory for other types of information(Frankland and Bontempi, 2005). For example, the ACC is activated after remote memory recall in a number of tasks (Bontempi
et al., 1999; Frankland et al., 2004; Maviel et al., 2004). Conversely, inactivation of the ACC disrupts recall of remote fivearm discrimination (Maviel et al., 2004) and contextual fear
(Frankland et al., 2004) memories, and lesions of the mPFC (thatinclude the ACC and prelimbic cortex) block remote (monthold) trace eye-blink memories (Takehara et al., 2003). The
present results extend this list to include a purely spatial memory;
one that requires the mouse to navigate to a fixed point in space
using an allocentric representation of a remotely learned
environment.
The exact nature of the role the ACC plays in remote memoryis less clear, however. One outstanding issue is whether the ACC
is important for memory storage or memory retrieval. The ACC
is a functionally heterogeneous structure, and activity in the
ACC is related to a number of cognitive [e.g., error detection,
effortful recall, attention (Carter et al., 1999; Miller, 2000)], motor [e.g., preparation and execution of movement (Paus, 2001)],
and sensory [e.g., pain (Price, 2000; Zhuo, 2005)] functions. Although a small proportion of cells within the dorsal ACC exhibit
place fields (Hok et al., 2005), the role of the ACC in remotememory appears to be conserved across tasks, regardless ofwhether they are spatial or nonspatial. The ACC is highly interconnected to other prefrontal regions, and is reciprocally connected to sensory, motor, and limbic cortices (Uylings et al.,
2003; Jones et al., 2005). This connectivity places the ACC in a
hub-like position, and therefore raises the possibility that thisregion coordinates retrieval of remote memories stored in distributed cortical networks.

References
Aggleton JP, Vann SD, Oswald CJ, Good M (2000) Identifying cortical inputs to the rat hippocampus that subserve allocentric spatial processes: asimple problem with a complex answer. Hippocampus 10:466 – 474.
Anagnostaras SG, Maren S, Fanselow MS (1999) Temporally graded retrograde amnesia of contextual fear after hippocampal damage in rats:
within-subjects examination. J Neurosci 19:1106 –1114.
Balogh SA, Radcliffe RA, Logue SF, Wehner JM (2002) Contextual and cued
fear conditioning in C57BL/6J and DBA/2J mice: context discriminationand the effects of retention interval. Behav Neurosci 116:947–957.
Bolhuis JJ, Stewart CA, Forrest EM (1994) Retrograde amnesia and memory
reactivation in rats with ibotenate lesions to the hippocampus or subiculum. Q J Exp Psychol B 47:129 –150.
Bontempi B, Laurent-Demir C, Destrade C, Jaffard R (1999) Timedependent reorganization of brain circuitry underlying long-term memory storage. Nature 400:671– 675.
Broadbent NJ, Squire LR, Clark RE (2006) Reversible hippocampal lesions

J. Neurosci., July 19, 2006 • 26(29):7555–7564 • 7563
disrupt water maze performance during both recent and remote memory
tests. Learn Mem 13:187–191.
Burgess N, Maguire EA, O’Keefe J (2002) The human hippocampus andspatial and episodic memory. Neuron 35:625– 641.
Carter CS, Botvinick MM, Cohen JD (1999) The contribution of the anterior cingulate cortex to executive processes in cognition. Rev Neurosci
10:49 –57.
Cave CB, Squire LR (1991) Equivalent impairment of spatial and nonspatial
memory following damage to the human hippocampus. Hippocampus
1:329 –340.
Clark RE, Broadbent NJ, Squire LR (2005a) Hippocampus and remote spatial memory in rats. Hippocampus 15:260 –272.
Clark RE, Broadbent NJ, Squire LR (2005b) Impaired remote spatial memory after hippocampal lesions despite extensive training beginning earlyin life. Hippocampus 15:340 –346.
Crawley JN, Belknap JK, Collins A, Crabbe JC, Frankel W, Henderson N,
Hitzemann RJ, Maxson SC, Miner LL, Silva AJ, Wehner JM, WynshawBoris A, Paylor R (1997) Behavioral phenotypes of inbred mousestrains: implications and recommendations for molecular studies. Psychopharmacology (Berl) 132:107–124.
Cui Z, Wang H, Tan Y, Zaia KA, Zhang S, Tsien JZ (2004) Inducible andreversible NR1 knockout reveals crucial role of the NMDA receptor inpreserving remote memories in the brain. Neuron 41:781–793.
Day M, Langston R, Morris RG (2003) Glutamate-receptor-mediated encoding and retrieval of paired-associate learning. Nature 424:205–209.
de Hoz L, Martin SJ, Morris RG (2004) Forgetting, reminding, and remembering: the retrieval of lost spatial memory. PLoS Biol 2:E225.
Eichenbaum H, Stewart C, Morris RG (1990) Hippocampal representationin place learning. J Neurosci 10:3531–3542.
Frankland PW, Bontempi B (2005) The organization of recent and remotememories. Nat Rev Neurosci 6:119 –130.
Frankland PW, Bontempi B, Talton LE, Kaczmarek L, Silva AJ (2004) The
involvement of the anterior cingulate cortex in remote contextual fear
memory. Science 304:881– 883.
Gusev PA, Cui C, Alkon DL, Gubin AN (2005) Topography of Arc/Arg3.1
mRNA expression in the dorsal and ventral hippocampus induced byrecent and remote spatial memory recall: dissociation of CA3 and CA1
activation. J Neurosci 25:9384 –9397.
Hoh TE, Kolb B, Eppel A, Vanderwolf CH, Cain DP (2003) Role of the
neocortex in the water maze task in the rat: a detailed behavioral and
Golgi-Cox analysis. Behav Brain Res 138:81–94.
Hok V, Save E, Lenck-Santini PP, Poucet B (2005) Coding for spatial goals
in the prelimbic/infralimbic area of the rat frontal cortex. Proc Natl Acad
Sci USA 102:4602– 4607.
Jones BF, Groenewegen HJ, Witter MP (2005) Intrinsic connections of the
cingulate cortex in the rat suggest the existence of multiple functionallysegregated networks. Neuroscience 133:193–207.
Kaczmarek L, Robertson HA (2002) Handbook of chemical neuroanatomy.
Amsterdam: Elsevier.
Kim JJ, Fanselow MS (1992) Modality-specific retrograde amnesia of fear.
Science 256:675– 677.
Leutgeb S, Leutgeb JK, Moser MB, Moser EI (2005) Place cells, spatial maps
and the population code for memory. Curr Opin Neurobiol 15:738 –746.
Maguire EA, Burke T, Phillips J, Staunton H (1996) Topographical disorientation following unilateral temporal lobe lesions in humans. Neuropsychologia 34:993–1001.
Martin SJ, de Hoz L, Morris RG (2005) Retrograde amnesia: neither partial
nor complete hippocampal lesions in rats result in preferential sparing of
remote spatial memory, even after reminding. Neuropsychologia
43:609 – 624.
Maviel T, Durkin TP, Menzaghi F, Bontempi B (2004) Sites of neocorticalreorganization critical for remote spatial memory. Science 305:96 –99.
Micheau J, Riedel G, Roloff EL, Inglis J, Morris RG (2004) Reversible hippocampal inactivation partially dissociates how and where to search in thewater maze. Behav Neurosci 118:1022–1032.
Miller EK (2000) The prefrontal cortex and cognitive control. Nat Rev Neurosci 1:59 – 65.
Morris RG, Garrud P, Rawlins JN, O’Keefe J (1982) Place navigation impaired in rats with hippocampal lesions. Nature 297:681– 683.
Morris RG, Moser EI, Riedel G, Martin SJ, Sandin J, Day M, O’Carroll C(2003) Elements of a neurobiological theory of the hippocampus: the7564 • J. Neurosci., July 19, 2006 • 26(29):7555–7564
role of activity-dependent synaptic plasticity in memory. Philos Trans R
Soc Lond B Biol Sci 358:773–786.
Moscovitch M, Rosenbaum RS, Gilboa A, Addis DR, Westmacott R, Grady C,
McAndrews MP, Levine B, Black S, Winocur G, Nadel L (2005) Functional neuroanatomy of remote episodic, semantic and spatial memory: aunified account based on multiple trace theory. J Anat 207:35– 66.
Moscovitch M, Nadel L, Winocur G, Gilboa A, Rosenbaum RS (2006) The
cognitive neuroscience of remote episodic, semantic and spatial memory.
Curr Opin Neurobiol 16:179 –190.
Moser E, Moser MB, Andersen P (1993) Spatial learning impairment parallels the magnitude of dorsal hippocampal lesions, but is hardly present
following ventral lesions. J Neurosci 13:3916 –3925.
Moser MB, Moser EI (1998) Distributed encoding and retrieval of spatial
memory in the hippocampus. J Neurosci 18:7535–7542.
Mumby DG, Astur RS, Weisend MP, Sutherland RJ (1999) Retrograde amnesia and selective damage to the hippocampal formation: memory forplaces and object discriminations. Behav Brain Res 106:97–107.
Nakazawa K, McHugh TJ, Wilson MA, Tonegawa S (2004) NMDA receptors, place cells and hippocampal spatial memory. Nat Rev Neurosci
5:361–372.
O’Keefe JM, Nadel L (1978) The hippocampus as a cognitive map. Oxford:
Oxford UP.
Paus T (2001) Primate anterior cingulate cortex: where motor control, driveand cognition interface. Nat Rev Neurosci 2:417– 424.
Paxinos G, Franklin KBJ (2000) The mouse brain in stereotaxic coordinates,
Ed 2. San Diego: Academic.
Price DD (2000) Psychological and neural mechanisms of the affective dimension of pain. Science 288:1769 –1772.
Remondes M, Schuman EM (2004) Role for a cortical input to hippocampal
area CA1 in the consolidation of a long-term memory. Nature
431:699 –703.
Riedel G, Micheau J, Lam AG, Roloff E, Martin SJ, Bridge H, Hoz L, PoeschelB, McCulloch J, Morris RG (1999) Reversible neural inactivation reveals
hippocampal participation in several memory processes. Nat Neurosci
2:898 –905.
Rosenbaum RS, Priselac S, Kohler S, Black SE, Gao F, Nadel L, Moscovitch M(2000) Remote spatial memory in an amnesic person with extensive bilateral hippocampal lesions. Nat Neurosci 3:1044 –1048.
Rosenbaum RS, Ziegler M, Winocur G, Grady CL, Moscovitch M (2004) “Ihave often walked down this street before”: fMRI studies on the hippocampus and other structures during mental navigation of an old environment. Hippocampus 14:826.

Teixeira et al. • The Anterior Cingulate Cortex and Spatial Memory
Sandkuhler J, Maisch B, Zimmermann M (1987) The use of local anaesthetic microinjections to identify central pathways: a quantitative evaluation of the time course and extent of the neuronal block. Exp Brain Res
68:168 –178.
Smith ML, Milner B (1981) The role of the right hippocampus in the recallof spatial location. Neuropsychologia 19:781–793.
Squire LR, Alvarez P (1995) Retrograde amnesia and memory consolidation: a neurobiological perspective. Curr Opin Neurobiol 5:169 –177.
Squire LR, Clark RE, Knowlton BJ (2001) Retrograde amnesia. Hippocampus 11:50 –55.
Steffenach HA, Witter M, Moser MB, Moser EI (2005) Spatial memory in
the rat requires the dorsolateral band of the entorhinal cortex. Neuron
45:301–313.
Sutherland RJ, Weisend MP, Mumby D, Astur RS, Hanlon FM, Koerner A,
Thomas MJ, Wu Y, Moses SN, Cole C, Hamilton DA, Hoesing JM (2001)
Retrograde amnesia after hippocampal damage: recent vs. remote memories in two tasks. Hippocampus 11:27– 42.
Takehara K, Kawahara S, Kirino Y (2003) Time-dependent reorganizationof the brain components underlying memory retention in trace eyeblinkconditioning. J Neurosci 23:9897–9905.
Teng E, Squire LR (1999) Memory for places learned long ago is intact after
hippocampal damage. Nature 400:675– 677.
Uylings HB, Groenewegen HJ, Kolb B (2003) Do rats have a prefrontal cortex? Behav Brain Res 146:3–17.
Wang H, Shimizu E, Tang YP, Cho M, Kyin M, Zuo W, Robinson DA, Alaimo
PJ, Zhang C, Morimoto H, Zhuo M, Feng R, Shokat KM, Tsien JZ (2003)
Inducible protein knockout reveals temporal requirement of CaMKII
reactivation for memory consolidation in the brain. Proc Natl Acad Sci
USA 100:4287– 4292.
Warburton EC, Aggleton JP, Muir JL (1998) Comparing the effects of selective cingulate cortex lesions and cingulum bundle lesions on water maze
performance by rats. Eur J Neurosci 10:622– 634.
Winocur G, Moscovitch M, Fogel S, Rosenbaum RS, Sekeres M (2005) Preserved spatial memory after hippocampal lesions: effects of extensive experience in a complex environment. Nat Neurosci 8:273–275.
Wolfer DP, Stagljar-Bozicevic M, Errington ML, Lipp HP (1998) Spatial
memory and learning in transgenic mice: fact or artifact? News Physiol Sci
13:118 –123.
Zhuo M (2005) Targeting central plasticity: a new direction of finding painkillers. Curr Pharm Des 11:2797–2807.

Journal of Neuroscience Supplemental Material
Involvement of the anterior cingulate cortex in the expression of remotespatial memory
Cátia M. Teixeira, Stephen R. Pomedli, Hamid R. Maei, Nohjin Kee & Paul W.
Frankland
Results
Inactivation of the ACC disrupts expression of remote spatial memory in C57B6
mice
We also examined the effects of lidocaine-induced inactivation of the ACC anddHPC in C57B6 mice. C57B6 mice require more training to acquire the same level of
performance as C57B6/129 hybrid mice in the water maze (Owen et al., 1997).
Therefore, to equate levels of performance in the probe tests, the C57B6 mice in thisstudy were trained for 7 (rather than 3) days. Over the course of training, mice in the PBS(F(6,234) = 40.19; p < 0.001), ACC (F(6,90) = 24.68; p < 0.001) and dHPC (F(6,84) =13.44; p < 0.001) groups all learned to find the platform with progressively shorter escapelatencies, and there were no differences in escape latencies between groups (nosignificant main effect of group [F(2,68) < 1] or group × training day interaction[F(12,408) = 1.33, p = 020]) (data not shown). This amount of training produced astrong, stable spatial memory in C57B6 mice: PBS-treated mice searched selectively inthe target zone (compared to other zones) in both the recent and remote probe tests(significant main effect of zone: F(1,40) = 29.58; p < 0.001) (Figure 1a). The
performance was quantitatively similar to that observed in the C57B6/129 hybrid micetreated with PBS in our previous experiment. Lidocaine inactivation of the ACC
preferentially disrupted performance in the remote, but not the recent, probe test (Figure1b). While the group × zone interaction was not significant (F(1,15) = 4.29, p = 0.056),
planned comparisons indicated that these mice spent more time searching the target zone(compared to other zones) only in the recent probe test (p < 0.05). Furthermore, the timespent searching the target zone in the recent probe test was also significantly more than in
the remote probe test (p < 0.05). Lidocaine infusions into the dHPC disrupted
performance in both the recent and the remote probe tests (Figure 1c). ANOVA on the1

probe test data for these mice revealed no significant main effects (group: F(1,14) = 2.26,
p = 0.16; zone F(1,14) < 1) nor group × zone interaction (F(1,14) = 2.13, p = 0.17).
Planned comparisons indicated that mice receiving lidocaine infusions into the dHPC did
not search selectively (target zone vs. other zones) in either the recent or the remote test(p > 0.05). Together these results show that the ACC is specifically involved in recall of
remote spatial memory, and they replicate the exact pattern of results in an independentgroup of mice in a different genetic background.

2

Figure 1. Effects of lidocaine inactivation of ACC and dHPC on recent and remotespatial memory in C57B6 mice. Spatial memory was evaluated in a probe test either 1day (recent group) or 30 days (remote group) following 7 days of training in the hidden
version of the water maze. The percent time spent searching the target zone (T) comparedto other (O) zones in probe tests is shown for mice that received infusions of either PBS(a) or lidocaine into the ACC (b) or dHPC (c).

3

References
Owen EH, Logue SF, Rasmussen DL, Wehner JM (1997) Assessment of learning by the
Morris water task and fear conditioning in inbred mouse strains and F1 hybrids:
implications of genetic background for single gene mutations and quantitative
trait loci analyses. Neuroscience 80:1087-1099.

4