Behavioral Neuroscience Copyright 2006 by the American Psychological Association

2006, Vol. 120, No. 3, 641– 650 0735-7044/06/$12.00 DOI: 10.1037/0735-7044.120.3.641

Hippocampal and Caudate Metabolic Activity Associated With Different

Navigational Strategies

Rubén Miranda, Eduardo Blanco, and Sandra Rubio

Azucena Begega Madrid Autonomous University
University of Oviedo

Jorge L. Arias
University of Oviedo

Hippocampal and striatal systems are widely related to spatial tasks. Depending on the strategies used,
different memory systems can be activated. In this study, the authors used the cytochrome c-oxidase
technique as a functional marker of the hippocampal and dorsal striatum activity related to training in
several water maze tasks. Current results show a differential participation of the hippocampal and striatal
systems in navigation. When spatial information is relevant, participation of the hippocampal system is
more important, and when the task is similar to a response learning one, the striatal system is more active.
According to computational models, CA3 seems to be more active when the associative demand is
higher, whereas CA1 and dentate gyrus activity are higher when spatial information processing is
required.

Keywords: spatial learning, Morris water maze, hippocampus, striatum, cytochrome c-oxidase

The hippocampus is a structure involved in memory processes
and learning tasks, such as the spatial ones, that require an elab-
orated processing of the incoming information (Eichenbaum,
Stewart, & Morris, 1990). In contrast, the striatum appears to be
specialized in response learning, which is a slower form of learn-
ing based on stimulus–response associations (White & McDonald,
2002).
Previous studies have shown that hippocampal and striatal sys-
tems can process information in parallel during a spatial task. Most
of the evidence suggesting a functional dissociation between hip-
pocampal and striatum systems comes from studies involving
lesions or pharmacological manipulations (Compton, 2004;
Eichenbaum et al., 1990; McDonald & White, 1994; Morris,
Garrud, Rawlings, & O’Keefe, 1982; Okaichi, 2001; Packard,
Cahill, & McGaugh, 1994; Packard & McGaugh, 1992, 1996;
Schroeder, Wingard, & Packard, 2002). These systems can inter-
act, and the nature of this interaction seems to be competitive
because hippocampal lesions or reversible inactivation improve
the performance on the basis of the striatum-dependent, cue-
Rubén Miranda, Eduardo Blanco, Azucena Begega, and Jorge L. Arias,
Laboratory of Psychobiology, School of Psychology, University of Oviedo,
Oviedo, Spain; Sandra Rubio, Psychobiology Area, School of Psychology,
Madrid Autonomous University, Madrid, Spain.
This research was sponsored by Ministerio de Educación y Ciencia
Grant SEJ2004-07445/PSIC. We thank Angel Nistal for her expert tech-
nical support inside the imaging analysis facility of the University of
Oviedo and Luis J. Santı́n for his helpful comments and critical reading of
this article.
Correspondence concerning this article should be addressed to Rubén
Miranda, who is now at the Department of Psychology, University of the
Balearic Islands, Ed. Beatriu de Pinós (Room 11), Carretera de Valldemossa
km 7.5, 07122 Palma de Mallorca, Spain. E-mail: ruben.miranda@uib.es
guided system (Compton, 2004; McDonald & White, 1994; Okai-
chi, 2001; Schroeder et al., 2002). Nevertheless, as Hamilton,
Rosenfelt, and Whishaw (2004) noted, some neurobiological ma-
nipulations may induce a bias in strategy preference leading to a
misinterpretation of the conclusions relating to the interaction
between brain function and behavioral outcomes.
Hippocampal or fimbria-fornix lesioned rats can solve spatial
tasks using nonspatial strategies (e.g., taxon strategies and dead
reckoning; Pouzet, Zhang, Feldon, & Rawlins, 2002) apparently
on the basis of an extrahippocampal navigation system (Alyan,
Jander, & Best, 2000; Whishaw, Hines, & Wallace, 2001). This
suggests that involvement of the hippocampus in spatial learning
depends on the task and the strategies used. In this way, when a
task can be solved by nonspatial strategies, the performance may
depend on other structures such as the striatum, as demonstrated in
response learning paradigms (Packard, 1999; Packard & Mc-
Gaugh, 1996; White & McDonald, 2002). Furthermore, given the
functional heterogeneity of the hippocampal divisions (Gilbert &
Kesner, 2003; Gilbert, Kesner, & DeCoteau, 1998; Gilbert,
Kesner, & Lee, 2001; Kesner, Gilbert, & Wallenstein, 2000; Lis-
man, 1999), a differential involvement of CA1, CA3, and dentate
gyrus (DG) probably occurs according to the different demands of
the tasks or depending on the navigation strategies used. For
instance, spatial learning impairments have previously been related
to specific deficits in spatial and temporal pattern separation pro-
cesses (Eichenbaum et al., 1990) that have been associated with
CA1 and DG functioning, respectively (Kesner et al., 2000).
Although in normal conditions (i.e., natural complex environ-
ments), rats seem to prefer distal landmarks for guiding their
behavior in familiar and well-lighted contexts (Whishaw et al.,
2001), it is also possible that more than one strategy is being used
simultaneously for more efficient navigation (Cain, Beiko, &
Boon, 1997). Likewise, multiple brain memory systems can be

641
642 MIRANDA, BLANCO, BEGEGA, RUBIO, AND ARIAS
simultaneously activated to process the different types of informa-
tion relevant for navigation that may determine a particular behav-
ioral output (i.e., navigation strategy). Some controversy exists
about which cues are preferred during navigation when multiple
sources of information are relevant simultaneously. Recently,
Okaichi (2001) proposed that a hierarchy in the use of problem-
solving strategies exists by suggesting that place strategy is the
favorite one followed by cue strategy and, finally, response strat-
egy. Nevertheless, Hamilton et al. (2004) and Baldi, Lorenzini, and
Bucherelli (2003) demonstrated that rats can use a combination of
local and procedural strategies. These results indicate that, in
conditions in which different cues (i.e., distal and proximal) exist,
rats can use both sources of information, which displays a com-
bination of strategies. This implies cooperation between cognitive
strategies that may require a cooperative functional interaction
between brain memory systems.
In this work, behavioral analysis of the strategies displayed in
the different water maze tasks was done in an attempt to clarify the
previous controversy. After delineating the representational or
associative demands of the tasks, we analyzed the oxidative me-
tabolism of the hippocampal and striatal systems, trying to better
understand the functional interactions between brain memory sys-
tems that control spatial abilities. Because the participation of
different brain areas in solving navigation problems can change
when any of them are not functioning (Sutherland & Hamilton,
2004), functional studies can provide interesting and useful infor-
mation for describing relationships among multiple memory sys-
tems during normal cognitive processes in the intact brain (Co-
lombo, Brightwell, & Countryman, 2003). Considering this, from
a noninvasive approach, we decided to test the value of cyto-
chrome c-oxidase (CO) quantitative histochemistry as a potential
functional marker to reveal specific changes in activity of different
brain areas correlated with the gradual acquisition of complex
tasks. CO is a key enzyme in cellular oxidative metabolism be-
cause it participates in the mitochondrial electron transport chain
involved in the direct production of energy as ATP that uses most
of the available oxygen. Local CO activity is considered a reliable
marker of brain oxidative metabolic potential because of the close
relationship between energy metabolism and neuronal activity
(Wong-Riley, 1989). In contrast to other metabolic markers, CO
activity can be used as an index of the sustained energy require-
ments of nervous tissue associated with brain function (Gonzalez-
Lima & Cada, 1994; Gonzalez-Lima & Jones, 1994). Moreover,
this technique has been shown to be sufficiently sensitive in
detecting changes in local cerebral oxidative metabolism induced
after associative learning (Nair, Berndt, Barrett, & Gonzalez-Lima,
2001; Poremba, Jones, & Gonzalez-Lima, 1998) and has been used
to assess functional changes correlated with spatial reference (Vil-
lareal, Gonzalez-Lima, Berndt, & Barea-Rodriguez, 2002) and
working memory (Conejo, Gonzalez-Pardo, Vallejo, & Arias,
2004).
Method
Rats and Groups
A total of 65 adult Wistar rats (approximately 90 days old) that weighed
300 ⫹/⫺ 50 g were obtained from the University of Oviedo’s central
vivarium. They were housed under standard conditions (12-hr light– dark
cycle with lights on from 08.00 to 20.00) at a constant room temperature
of 21 ⫹/⫺ 2 °C with ad lib access to food and water. All experimental
procedures conducted on the rats were approved by a local veterinary
committee from the University of Oviedo’s vivarium, and subsequent
handling strictly followed the European Communities Council’s Directive
86/609 and the American Psychological Association’s ethical code.
Rats were assigned randomly to the different groups that corresponded
with the different training and control conditions: (a) the hidden platform
(HP) group (Group 1), which performed the HP task (n ⫽ 15 water maze,
n ⫽ 9 cytochrome); (b) the visual fixed (VF) group (Group 2), which
performed the VF task (n ⫽ 10 water maze, n ⫽ 9 cytochrome); (c) the
visual moving (VM) group (Group 3), which performed the VM task (n ⫽
15 water maze, n ⫽ 10 cytochrome); (d) the swimming control (SC) group
(Group 4; n ⫽ 15 water maze, n ⫽ 10 cytochrome); and (e) the caged
control (CC) group (Group 5; n ⫽ 10 cytochrome).
Rats submitted to a water maze task were assigned pseudorandomly to
CO histochemistry groups. Only brain samples in optimal conditions for
densitometric analysis were included in the study.
Water Maze
We trained the rats in several Morris water maze escape tasks (Morris,
1981, 1984) using a 1.5-m-diameter black fiberglass pool, which was 75
cm high and 50 cm above the floor. The pool was filled with tap water to
a height of 32 cm, and a black escape platform was placed 2 cm beneath
the water surface. The water temperature was kept at 21 °C during the
entire test period. The experimental room had numerous visual cues (such
as colored maps, posters, and plastic dishes fixed on the walls), a shelf,
covered windows, and a table. Lighting was provided by two halogen
spotlights (500 W) placed on the floor and facing the walls. We recorded
all swim paths of the rats live and later analyzed them using a computerized
video-tracking system (Smart, Panlab, Spain).
HP Task
The entire protocol of the HP task took 5 days. On Day 1, rats received
two habituation trials 60 s in duration with an intertrial interval of 30 s
remaining in a black plastic bucket. During these trials, rats were released
facing the pool wall from two different points and were allowed to freely
explore the water maze. After habituation (Days 2–5), we trained the rats
daily using a single six-trial session. In each trial, the rats were released
randomly from one of four start locations and had to search for a hidden
escape platform beneath the water surface. The platform was located in the
same position during the training days. Rats failing to find the platform
after 60 s were gently guided to it by hand, where they remained for 15 s
before being introduced into a black plastic bucket for 30 s. On Day 5 after
the last training trial, rats were submitted to a one-trial probe test in which
the platform was removed, and the rats were allowed to swim for 30 s.
VF Task
The general procedure for this task was the same as for the HP task,
which consisted of a habituation day, a training phase, and a probe test. In
this task, the escape platform was painted white and projected 2 cm above
the water surface. A cylindrical object (3 cm diameter/5 cm height) was put
onto the platform as a visual beacon. For each trial the position of the
platform was the same, and only the start point was changed.
VM Task
For this task, we used the same procedure previously described but
changed the position of the visual escape platform. Here the location of the
platform was changed for each trial to avoid rats using the distal landmarks
of the room.
 HIPPOCAMPAL AND CAUDATE METABOLIC ACTIVITY
SC Task
In this task, rats were placed in the water maze in the absence of an
escape platform, which allowed them to swim freely. The procedure was
conducted during the same number of trials and training days as in the HP
task. The duration of each trial coincided with the mean escape latencies
performed by the rats trained in the HP task.
Behavioral Analysis
During the training phase in each task, we recorded the distance covered
to reach the platform and the escape latencies. We performed statistical
analyses using the mean values of these parameters for each daily training
session. For analysis of the probe test performance, two different virtual
divisions of the pool were used: quadrant and ring divisions. First, the pool
was divided virtually into four quadrants (A, B, C, and D/escape), and
second, it was divided into concentric circular regions or rings with the
same area (border, Ring 2, Ring 3/platforms, and nucleus; see Figure 1).
On the basis of these divisions, the mean percentage of the time swam in
each sector was analyzed.
Histochemistry of the CO
Tissue Preparation
Rats from control and trained groups were deeply anesthetized by
intraperitoneal injection of sodium pentobarbital (70 mg/kg) 1 hr after
finishing the behavioral procedure. Rats were transcardially perfused with
a phosphate buffer solution (pH 7.4; 0.1 M). Their brains were quickly
removed and coronally sectioned to obtain thick slices extending from
optic chiasm to the caudal hypothalamic area according to Paxinos and
Watson’s (1986) atlas. Then, the slices were immersed in a 20% sucrose
buffer solution at 4 °C until sinking (approximately 12 hr). Tissue was
embedded in a cryoprotective gel (optimal cutting temperature compound;
Miles, Elkhart, Indiana) frozen in Freon-22 (chlorodifluoromethane,
CIF2CH) and stored at ⫺70 °C. Next, 20-␮m-thick sections from the brains
were obtained at ⫺20 °C with a cryostat microtome (Microm, Heidelberg,
Figure 1. Virtual divisions of the Morris water maze used for probe test analyses. Left: Quadrant division (A,
B, C, and D/escape). The escape platform was placed in Quadrant D in tasks with the platform in a fixed position
(i.e., hidden platform and visual fixed). Right: Ring division (border, Ring 2, Ring 3/platforms, and nucleus).
Escape platform was always placed in Ring 3.
643
Germany). These thin sections were put on glass slides previously cleaned
with alcohol (100%) and then stored at ⫺80 °C.
CO Histochemistry
Sections from the brains were used to perform CO histochemistry. We
used a modified version of the method originally described by Wong-Riley
(1979), which was based on the method developed by Sukekawa (1991). In
brief, slides were lightly fixed for 5 min with a 1.5% glutaraldehyde
solution, rinsed 3 times in phosphate buffer, and incubated at 37 °C for 2
hr in the dark and with continuous stirring in a solution that contained 50
mg 3,3⬘-diaminobenzidine, 15-mg cytochrome c (Sigma Chemical, St.
Louis, Missouri), 4 g sucrose per 100 ml phosphate buffer (pH 7.4; 0.1 M).
The slides were rinsed 3 times with phosphate buffer, dehydrated, and
coverslipped with Entellan (Merck, Whitehouse Station, New Jersey).
Quantitative Densitometric Analysis
We measured CO histochemical staining intensity by densitometric
analysis using a charge-coupled device digital camera (Leica DC-300; E
Licht Company, Denver, Colorado) coupled to a light microscope (Olym-
pus, Tokyo, Japan) and image analysis software (Leica Q-Win; E Licht
Company, Denver, Colorado). A minimum of six measurements, from 8
bits gray scale images, were taken in the dorsal striatum (i.e., caudate-
putamen) and CA1, CA3, and DG of the antero-dorsal hippocampus from
each rat and expressed as arbitrary units of optical density. Delimitation of
the hippocampus and striatum was performed according to Paxinos and
Watson’s (1986) atlas. Antero-posterior coordinates for the studied regions
were approximately as follows: from ⫺1.30 mm to ⫺2.80 mm for caudate-
putamen complex and from ⫺2.56 mm to 3.80 mm for hippocampus from
bregma. Similar to the procedure described by Nair and Gonzalez-Lima
(1999), to establish comparisons and count for possible staining variations
between brain sections from different staining baths, we normalized mea-
surements by means of a ratio between the values of the mean hippocampal
activity and subregion activity. To compare the activity measurements of
the striatum with the hippocampus, we used subtracted values.
644 MIRANDA, BLANCO, BEGEGA, RUBIO, AND ARIAS

Statistical Analysis
We analyzed escape latencies or path lengths in each trained group using
one-way repeated measures analyses of variance (ANOVAs), with training
day as the repeated-measures factor and group as the intersubjects factor.
Bonferroni’s post hoc tests were used to assess differences between days,
and Dunnett’s tests were used to assess differences between groups be-
cause the variances obtained were unequal. For the probe test performance,
two different analyses were made: (a) We performed intragroup analysis
using a one-way repeated measures ANOVA, with mean percentage time
spent by sector as the repeated-measures factor, and (b) we performed
intergroup analysis using a one-way ANOVA, with group as the intersub-
jects factor and mean percentage time spent in escape quadrant as the
dependent variable. Bonferroni’s post hoc tests were performed to assess
differences in time spent in each sector for each group and to assess
differences between groups in the time spent in Quadrant D (escape).
We analyzed CO activity differences between main hippocampal divi-
sions (CA1, CA3, and DG) in each group separately using repeated
measures ANOVAs. Posteriori pairwise comparisons were made to reveal
activity differences between regions. Group differences in CO-transformed
activity from each main hippocampal division were assessed by one-way
ANOVAs. Bonferroni’s post hoc tests were used to assess differences in
CO activity between means when ANOVAs indicated significant overall
differences.
Finally, one-way ANOVAs were used to assess differences in CO
hippocampal-striatum subtracted activity, and Bonferroni’s post hoc tests
were used to assess differences in CO activity between groups.
Results
Behavioral Tests
Escape Latencies
Mean escape latencies significantly decreased during training
days, F(3, 35) ⫽ 31.666, p ⬍ .001, which resulted in significant
differences between groups, F(2, 37) ⫽ 12.385, p ⬍ .001, with no
significant differences in the interaction between both factors, F(6,
70) ⫽ 1.076, ns. Bonferroni’s post hoc test showed statistical
significant differences between Day 1 and Days 2, 3, and 4, and
between Day 2 and Days 1, 3, and 4 ( p ⬍ .05). No significant
differences existed between Days 3 and 4. Dunnett’s post hoc test
revealed that HP rats’ latencies were significantly greater than
those of VF and VM groups ( p ⬍ .05) and that no differences
existed between VF and VM groups (see Figure 2).
Distance Covered
As shown in Figure 2, the mean path lengths measured signif-
icantly decreased during training days in all groups, F(3, 35) ⫽
27.809, p ⬍ .001. Group effect was significant, F(2, 37) ⫽ 17.035,
p ⬍ .001, but the interaction was not, F(6, 70) ⫽ 1.079, ns.
Bonferroni’s post hoc test revealed significant differences between
Day 1 and Days 2, 3, and 4, and between Day 2 and Days 1, 3, and
4 ( p ⬍ .05), which corresponded with the greater distances with
Days 1 and 2. The optimal performance was reached at the end of
Day 2 of training because no differences existed between Days 3
and 4. Dunnett’s post hoc test showed that the distance covered by
VF rats was significantly less than that covered by HP and VM rats
( p ⬍ .05).
Overall activity of control rats (SC) was similar to HP rats
because a one-way repeated measures ANOVA showed nonsig-
nificant differences in the mean distance covered between both
groups, F(1, 26) ⫽ 1.368, ns.
Probe Test
Quadrant division. Intragroup analyses for the mean percent-
age time spent in each quadrant of the pool showed statistically

40 1000
Mean distance covered (+/-SEM) (cm.)
Mean escape latency (+/- SEM) (sec.)

HP HP
35 VF VF
VM 800 VM
30

25 600

20

400
15

10
200
5

0 0
1 2 3 4 1 2 3 4

Days Days

Figure 2. Left: Mean escape latencies performed during training. Visual fixed (VF) and visual moving (VM)
groups displayed significantly lower latencies compared with the hidden platform (HP) group. All groups
reached asymptotic behavior at the end of the 2nd training day. There were significant differences between Day
1 compared with Days 2, 3, and 4, and between Day 2 compared with Days 1, 3, and 4, which corresponded to
the greater latencies with Days 1 and 2. Right: Mean distance covered during training. VF rats covered
significantly lower distances compared with HP and VM rats. All groups reached asymptotic behavior at the end
of the 2nd training day. Significant differences were found between Day 1 compared with Days 2, 3, and 4, and
between Day 2 compared with Days 1, 3, and 4. No differences were found between the HP and swimming
control groups. Error bars represent standard error of the means (SEMs).
 HIPPOCAMPAL AND CAUDATE METABOLIC ACTIVITY 645

significant differences in the HP group, F(3, 12) ⫽ 49.674, p ⬍
.001; VF group, F(3, 7) ⫽ 6.993, p ⫽ .016; and SC group, F(3,
12) ⫽ 8.705, p ⬍ .001. Rats trained in the VM task showed no
preference for swimming in any of the quadrants because no
significant differences were found, F(3, 12) ⫽ 1.118, ns.
Bonferroni’s post hoc analysis revealed that HP rats swam
significantly longer in Quadrant D (escape) compared with the rest
of the quadrants ( p ⬍ .05). Rats trained in the VF task spent more
time swimming in Quadrants A and D/escape compared with
Quadrants B and C ( p ⬍ .05). SC rats spent significantly less time
in swimming in Quadrant B compared with Quadrants A, C, and
D/escape ( p ⬍ .05; see Figure 3).
Intergroup analysis for the mean percentage time spent in Quad-
rant D (escape) revealed statistically significant differences be-
tween groups, F(3, 51) ⫽ 39.034, p ⬍ .001. Post hoc analysis
showed that rats trained in the HP task spent significantly more
time in the quadrant of the maze where the escape platform was
constantly located in the HP and VF tasks compared with the other
groups ( p ⬍ .05; see Figure 3).
Ring division. Intragroup analyses for the mean percentage
time spent in each circular sector of the pool showed statistically
significant differences in the HP group, F(3, 12) ⫽ 14.722, p ⬍
.001; VF group, F(3, 7) ⫽ 6.767, p ⫽ .018; VM group, F(3, 12) ⫽
17.399, p ⬍ .001; and SC group, F(3, 12) ⫽ 548.682, p ⬍ .001.
Post hoc analyses revealed that HP rats spent significantly more
time in Rings 2 and 3 compared with the nucleus ( p ⬍ .05). Rats
trained in the VF task swam significantly longer in Ring 2 com-
pared with the border ( p ⬍ .05). VM rats spent more time in Rings

70 QUAD A
QUAD B
Mean percentage time in quadrants

QUAD C
60
QUAD D (Escape)

50

40

30

20 * * *
* *
*
10

0
HP VF VM SC

Figure 3. Results of the probe test analyses that used the virtual quadrant division. Top: Representative
trajectories during the probe test of the hidden platform (HP) group (left), the visual moving (VM) group
(middle), and the swimming control (SC) group (right). Bottom: HP rats spent significantly more time
(percentage) in Quadrant D/escape compared with Quadrants A, C, and B. Visual fixed (VF) rats spent significantly
more time (percentage) in Quadrant D/escape compared with Quadrants B and C. No differences existed in the
time spent in the escape quadrant compared with Quadrant A/start. VM rats showed no differences in the time
spent in each quadrant. SC rats spent significantly less time in Quadrant B during the last training day. Group
comparison of the mean percentage time spent in Quadrant D/escape showed that the HP rats spent significantly
more time swimming in the escape quadrant (error bars represent standard error of the means; *p ⬍ .05).
646 MIRANDA, BLANCO, BEGEGA, RUBIO, AND ARIAS

2 and 3 compared with the nucleus ( p ⬍ .05). Control rats (SC)
spent significantly more time in the border compared with the rest
of the sectors ( p ⬍ .05; see Figure 4).
CO Activity
Hippocampal Activity
Intragroup analyses for CO activity differences among main
hippocampal divisions (CA1, CA3, and DG) showed statistically
significant differences in each group ( p ⬍ .001). Posterior pair-
wise comparisons revealed a higher CO activity in CA3 followed
by DG and CA1, with the lowest CO activity in the HP group ( p ⬍
.05). In the remaining groups, CO activity of CA1 was signifi-
cantly lower than CA3 and DG ( p ⬍ .05). No significant differ-
ences existed between CA3 and DG in these groups.
To assess group differences in CO activity of the main hip-
pocampal divisions, we performed one-way ANOVAs that com-
pared the mean CO activity that resulted from transforming CO
values into activity ratios. These ratios were established as follows:
hippocampal division CO activity (CA1, CA3, or DG)/mean hip-
pocampal CO activity.
ANOVAs showed statistically significant differences among
groups in CA1, F(4, 43) ⫽ 5.345, p ⫽ .001; CA3, F(4, 43) ⫽
4.694, p ⫽ .003; and DG, F(4, 43) ⫽ 3.708, p ⫽ .011. Post hoc
analyses revealed a lower activity in CA1 of SC rats compared
with VF and CC groups ( p ⬍ .05). In CA3, the VM group
presented a higher activity compared with the VF group ( p ⬍ .05).
Finally, the CO activity of DG in the SC and HP groups was higher
compared with the CC group ( p ⬍ .05; see Figure 5).
Hippocampal to Caudate Activity
To compare the CO activity of the hippocampal and striatal
among groups, we performed a one-way ANOVA with the mean
CO subtracted activity between hippocampus and striatum as the
dependent variable. Overall, statistically significant differences
were detected, F(4, 42) ⫽ 19.904, p ⬍ .001, and a Bonferroni’s
post hoc test revealed that the activity in the CC group was
significantly lower than the rest of the groups and that the activity
in the VM group was higher than the rest of the groups ( p ⬍ .05).
No significant differences were detected between the SC, VF, and
HP groups (see Figure 6).
Discussion
The main goal of the current work was to assess the functional
activity, by means of the CO quantitative histochemistry, of the
hippocampus and dorsal striatum related to different water maze
tasks involving multiple navigation strategies. The behavioral per-
formance of the rats was measured as distance covered and escape
latency. All rats mastered the tasks after 2 days of training (i.e., 12
trials), although VF rats displayed shorter latencies and trajectories
compared with HP rats, and VM rats displayed long trajectories
similar to HP rats with short latencies similar to VF rats (see
Figure 2). These results reflect that when proximal and distal cues
are available (i.e., VF task), rats can use both sources of informa-
tion to locate a place. It also seems that the presence of a proximal
cue can facilitate acquisition of the task. This effect was previously
reported by testing hippocampal rats in a water maze (Morris et al.,
1982) and in a radial maze (Ramos, 2002), suggesting a differen-
tial involvement of the hippocampal system in the standard water
maze task (i.e., HP) and in the visual version (i.e., VF). However,
the behavior displayed by VM rats suggests that these rats might
have been using another strategy as well as the cue-guided one. For
instance, according to the results of Baldi et al. (2003), they could
have used a procedural strategy based on the distance relationship
between the platform location and the wall of the pool. Moreover,
it is also possible that rats tried to use the distal landmarks as

80 Border (B)
Ring 2 (R2)
70 Ring 3 (R3)
Mean percentage time in rings

Nucleus (N)
60

50

40

30

20
HP: R2, R3 > N
VF: R2 > B
10
VM: R2, R3 > N
SC: B > R2 > R3 > N
0
HP VF VM SC

Figure 4. Results of the probe test analyses that used the virtual ring division. Hidden platform (HP) rats spent
significantly more time (percentage) swimming in Rings 2 and 3 compared with the nucleus. Visual fixed (VF)
rats spent significantly more time (percentage) in Ring 2 compared with the border. Visual moving (VM) rats
swam significantly more time in Rings 2 and 3 compared with the nucleus. The swimming control (SC) rats
showed significant differences in the mean percentage of time spent in each sector during the last training session
(error bars represent standard error of the means).
 HIPPOCAMPAL AND CAUDATE METABOLIC ACTIVITY 647

1.10
CC
SC
1.05 HP
VF
VM
CO activity ratio (a.u.)

1.00

0.95

0.90

0.85

0.80
CA1 CA3 DG

Figure 5. Cytochrome c-oxidase (CO) activity in hippocampal subfields. CA1: There were significant differ-
ences between the swimming control (SC) group and caged control (CC) and visual fixed (VF) groups. CA3: The
activity of the VF group was significantly lower compared with the visual moving (VM) group. Dentate gyrus
(DG): There were significant differences between the CC group compared with the SC and hidden platform (HP)
groups (error bars represent standard error of the means).

another source of information to navigate. Because the platform
position changed in each trial and also its relation to distal land-
marks, using this information, the rats’ initial trajectories would
have been mistaken and, after not finding the platform in its
previous location, they would have to correct the trajectory to
reach the new platform’s location on the basis of the proximal cue
(i.e., visible platform). This strategy selection, recently reported by
Hamilton et al. (2004), causes rats to take longer and less accurate
trajectories that cover greater distances, which possibly explains
the differences compared with the VF group. However, from the
sole analysis of the distance covered, we cannot confirm or disre-
gard that rats are using this kind of place strategy involving spatial
working memory.
Probe test analyses confirmed that HP rats preferentially swam
in the escape quadrant, indicating that they presumably were using
distal landmarks and a spatial strategy to navigate. However, rats
trained in the VM task, in the absence of the visible platform,
showed no preference for any quadrant and displayed a circular
swimming behavior similar to SC rats (see Figure 3). This indi-
cates that in the VM task, the distal landmarks of the room were
not important to identify a target place defined by quadrants;
however, this does not necessarily imply that no spatial relation-
ships were established between room landmarks and different
water maze locations. With the aid of the virtual ring division of
the pool, we observed that all groups trained with the escape
platform (i.e., HP, VF, and VM) in the probe trial spent nearly 60%
of the time swimming inside Rings 2 and 3 (platforms ring),
whereas the SC group remained swimming in the border area for
approximately 75% of time during the last training session (see
Figure 4). These data reflect that VM rats, in contrast to SC rats,
acquired some knowledge about the constant relationship between
the platform locations and the walls. Finally, VF rats during the
probe test showed a preference for swimming in two quadrants of
the pool (start and escape). According to a recent report by
Hamilton et al. (2004), these results suggest that these rats prob-
ably used a combination of place and cue strategies during the
training and probe test. If these rats were only using a place
strategy during training, probably due to overshadowing of the
intramaze cues (Chamizo, Sterio, & Mackintosh, 1985), then the
results of the probe test should have been similar to the HP group,
swimming preferentially only in the escape quadrant. However,
they swam a similar time in both escape and start quadrants, and
group comparisons showed that the HP rats swam significantly
longer in the escape quadrant. In comparison, if these rats were
navigating using only a cue strategy, probably due to a reduction
in the attention and associability of the spatial cues derived from
the habituation phase, then they would have shown no preference
for any quadrant, as occurred with the VM group (see Figure 3).
Together, the results of the behavioral analyses show that rats use
different navigation strategies depending on the water maze train-
ing. When rats are trained to find a hidden platform and distal cues
are available, the rats preferentially use a local strategy. If rats
have to reach a visible platform placed at a fixed location, they use
a combination of place and cue-guided strategies. Finally, if the
visible platform is placed at different locations and at a specific
distance from the wall of the pool, then the rats use a cue-guided
648 MIRANDA, BLANCO, BEGEGA, RUBIO, AND ARIAS
50
CC
SC
HP
40
CO substracted activity (a.u.)
VF
VM
30
20 *
10
Figure 6. Comparison of the cytochrome c-oxidase (CO) activity of the striatum relative to the hippocampus.
The caged control (CC) group showed significantly lower activity compared with all groups, and visual moving
(VM) rats showed significantly higher activity also compared with all groups. No significant differences were
found between the swimming control (SC), hidden platform (HP), and visual fixed (VF) groups (error bars
represent standard error of the means; *p ⬍ .05).
strategy combined with a procedural strategy. In general, it seems
that rats, instead of using only one strategy as the best one, can
combine several strategies that attend to all the available informa-
tion to achieve a more efficient navigation. Taking this into ac-
count, in this work we decided to study brain functional correlates
between the different tasks and their demands with the activity of
the hippocampal and striatal systems. For this purpose, we used
CO quantitative histochemistry as a potential functional marker,
which is useful to reveal changes in brain oxidative metabolism
induced by sustained energy requirements derived from the grad-
ual acquisition of complex (water maze) tasks (Gonzalez-Lima &
Cada, 1994; Gonzalez-Lima & Jones, 1994; Wong-Riley, 1979,
1989).
The posttraining functional study revealed a putative differential
participation of hippocampal main divisions and dorsal striatum in
the water maze tasks. CO activity of CA1 and DG was signifi-
cantly higher in VF and HP groups, respectively, whereas CA3
activity was higher in the VM group (see Figure 5). As discussed
previously, VF and HP groups can be considered as place learners,
and the VM group demonstrated a navigation based on a cue
strategy, although they also exhibited acquisition of procedural
skills (i.e., circular swimming at a specific distance from the wall
of the pool) that may have involved some kind of intramaze
allothesis (Baldi et al., 2003). Hence, it cannot be disregarded that
these rats have not learned the task completely independently of
spatial processing; indeed, spatial working memory can also be
involved.
Spatial and temporal pattern separation processes have been
reported to be essential to solve spatial tasks, and a failure in these
functions can explain spatial learning deficits (Eichenbaum et al.,
1990; Kesner et al., 2000). Our results confirm that CA1 and DG
have an important role in the acquisition of water maze tasks
requiring spatial processing. However, the increased activity of
*
CA3 after VM training can be related to a spatial working memory
processing or more likely with a prolonged associative demand
during training. Because in this task the platform location, as well
as the release points, varied in each trial, possibly new associations
between the visible platform, spatial landmarks, and motor re-
sponses would be expected to be formed in each trial. However, it
appears that the mere activation of one hippocampal subfield is not
sufficient to explain a task-dependent processing. For example, SC
rats submitted to a simple forced swimming task presented an
unforeseen high activity in DG, similar to HP rats, but both groups
clearly differed in the CA3 and CA1 levels of activity (see Figure
5). Conversely, HP and VF rats presented a similar pattern of
activity with low CO activity in CA3 and high CO activity in CA1
and DG. Nevertheless, from our data, it is unclear which kind of
demand can alter hippocampal CO levels in SC rats, as it is
possible that the increase in CO activity observed in DG reflects a
spatial processing of the room containing the water maze.
Cumulative evidence has suggested that the hippocampus is
involved in multiple memory types (Squire, 1992) and has empha-
sized its important role in spatial learning (Eichenbaum et al.,
1990; Morris et al., 1982; O’Keefe & Nadel, 1978). Nevertheless,
it is becoming clear that spatial learning may not depend exclu-
sively on the hippocampus because rats with hippocampal lesions
can solve spatial tasks using nonspatial strategies (Alyan et al.,
2000; Pearce, Roberts, & Good, 1998; Pouzet et al., 2002;
Whishaw et al., 2001). These findings suggest that multiple brain
systems interact during normal navigation and that a relationship
exists between the systems’ activity and behavioral strategies. The
striatum is continuously associated with cue navigation and re-
sponse learning, and many studies have reported functional disso-
ciations and interactions between hippocampal and striatal activity
on navigation (Colombo et al., 2003; Compton, 2004; McDonald
& White, 1994; Mizumori, Yeshenko, Gill, & Davis, 2004; Okai-
 HIPPOCAMPAL AND CAUDATE METABOLIC ACTIVITY
chi, 2001; Packard, 1999; Packard & McGaugh, 1992, 1996;
Schroeder et al., 2002; White & McDonald, 2002).
In our work, we have introduced a novel functional approach to
determine in which training or control condition either the striatum
or the hippocampus has a greater participation. All groups that
were submitted to a water maze task presented an increase in CO
activity in the striatum (relative to the hippocampus) compared
with the CC group. This increase was especially higher in the VM
group, suggesting that cue/response learning involves a greater
energy demand for the striatum than for the hippocampus (see
Figure 6). Although it is well known that the striatum plays a key
role in motor activity, it is unlikely that our results can be ex-
plained by a specific greater locomotor activity or motor experi-
ence in the VM rats. During water maze training, the longer escape
latencies and distances covered corresponded to the SC group that
presented low CO activity levels, similar to HP and VF rats.
Furthermore, it has been reported that a long period of physical
exercise (6 months) is required to observe a motor activity-related
increase in the CO levels of the striatum (McCloskey, Adamo, &
Anderson, 2001).
In summary, our results indicate that rats may use as much
information as they can to find an escape platform by combining
more than one navigation strategy. Hippocampal and striatal sys-
tems can also be activated in parallel, suggesting a functional
participation of both systems to solve spatial problems with dif-
ferent cognitive demands. CO histochemistry appears to be suffi-
ciently sensitive to detect changes in prolonged neuronal activity
as a consequence of relatively more complex tasks compared with
conditioning paradigms in which this technique was used previ-
ously (Nair et al., 2001; Poremba et al., 1998). Our results are in
agreement with a previous functional study by Colombo et al.
(2003), which reported more phosphorylation of cAMP response
element binding protein and c-Fos expression in the hippocampus
and striatum of place and response learners, respectively. Here,
with the aid of the CO technique, we observed increased energy
metabolism in (a) hippocampal CA1 and DG, after the processing
of local landmarks underlying a place strategy as well as in (b)
hippocampal CA3 and dorsal striatum, following associative pro-
cessing of proximal (taxon) cues underlying a cue-guided or re-
sponse strategy. Further studies with functional markers such as
CO, with a focus on other cerebral regions, could be useful to
clarify the interaction between multiple brain memory systems
during spatial learning in intact brains.
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Received May 31, 2005
Revision received January 23, 2006
Accepted January 24, 2006 䡲