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Fos imaging reveals ageing-related changes in hippocampalresponse to radial maze discrimination testing in mice
Khalid Touzani,� Aline Marighetto and Robert JaffardCNRS, UMR-5106, Laboratory Neurosciences Cognitives, Avenue des Facultes, 33405 Talence Cedex, France
Keywords: cognitive ageing, declarative/ relational memory, immediate early gene, long-term retention, short-term retention
Abstract
A two-stage radial arm maze (RAM) task has been designed recently to demonstrate a specific age-related memory deficit in mice. Ithighlights the contrast between normal and deficient memory expression in a spatial discrimination problem depending on how to-be dis-criminated arms were presented to the animal. This specific deficit has been interpreted as a preferential loss in a relational/declarativeform of memory, thereby implying an underlying hippocampal dysfunction. To test this hypothesis, neuronal activation measured by Fosimmunostaining was compared in aged (21–23 months) and adult (4–6 months) mice trained in the aforementioned task and killed aftera retention session consisting in age-insensitive probe trials, performed 6 days later (6-day RAM). Two comparison conditions wereincluded: (i) repeated locomotor training on a treadmill (TM); (ii) the same RAM training, except for the use of a longer (30 days insteadof six) retention interval (30-day RAM). Although all RAM groups displayed similar levels of performance at the end of the experiment,immediately before the mice were killed, significant between-group differences in brain activation were observed. In adult mice, 6-dayRAM testing was associated with greater septal and hippocampal (CA1, CA3, DG) Fos expression than the TM condition. Lengtheningthe retention interval from 6 days to 30 days resulted in a significant decrease in RAM testing-induced Fos expression in most of the septo-hippocampal regions. With respect to adult mice, aged mice displayed reduced Fos expression (except for DG) and a lack of inter-relationships between levels of Fos produced in each of the SH regions, in the 6-day RAM testing condition. Conversely, there was noeffect of ageing on Fos expression associated with either TM training or 30-day RAM testing. These results are interpreted as reflectingage- (or time-) related alterations in recruiting of brain structures that underlie a relational/declarative form of memory expression.
Introduction
There is a general agreement in both human and animal experimenta-
tion that memory performance declines from early to late adulthood,
and that such age-related cognitive decline is more prominent and
severe in certain tasks (as reviewed by Gallagher & Rapp, 1997).
Neuroimaging studies in aged human subjects have revealed that
abnormal activity (either higher or lower than in younger adults) in
specific brain regions can be obtained depending on the specific task(s)
used (for a review, see D’Esposito, 1999; Grady & Craik, 2000). Given
the limited history of the application of functional neuroimaging
techniques in the study of cognitive decline associated with normal
ageing, continual effort in this direction is needed to further reveal the
complex functional anatomical changes associated with senescence.
Surprisingly, very few studies in animals (i.e. Nagahara & Handa,
1997; Zhang et al., 2000) have tackled this issue by performing
functional imaging experiments. Using mice as subjects, the present
work attempts just this.
Using a specifically designed two-stage behavioural testing proce-
dures in a radial arm maze (RAM), we have previously provided
evidence that both ageing (Marighetto et al., 1999, 2000) and hippo-
campal lesions in adult subjects (Etchamendy et al. in press) produced
the same pattern of spared vs. impaired performance depending only
on the way discrimanda (i.e. arms of the maze) were presented to the
animal (mouse). Namely, successful discrimination between arms of
opposing valence was observed when these arms were presented one at
a time (successive go-no-go discrimination) or all simultaneously (six-
choice discrimination). Yet such demonstrable acquired knowledge
failed to guide the same aged or hippocampal-lesioned mice towards
the positive arms when confronted with an explicit choice between two
of the same arms (two-choice discrimination). In line with similar
findings in rats (Eichenbaum et al., 1988, 1989), this pattern of deficit
was interpreted as reflecting a selective loss of relational memory
which, in aged animals, would stem from hippocampal physiological
dysfunctions that have been described in the ageing (reviewed in
Gallagher & Rapp, 1997).
The present study was designed to further examine this hypothesis
by comparing testing-induced activation of specific brain regions in
adult and aged mice using immunodetection of Fos protein. The
expression of the protein product of the proto-oncogene c-fos is
believed to be indicative of changes in neuronal activity (Morgan &
Curran, 1989; McCabe & Horn, 1994) and has repeatedly been shown
to be induced under learning conditions (e.g. Herdegen & Leah, 1998;
Tischmeyer & Grimm, 1999; Wan et al., 1999; Vann et al., 2000)
although intense Fos labelling might not by itself represent an index of
functionality of the brain regions considered (see Passino et al., 2002).
In the present experiment we tried to circumvent several potential
‘false positive’ age-related differences in testing-induced brain acti-
vity. First, in most of the experiments so far performed (but see Bennett
et al., 2001) one cannot disambiguate brain activation differences
related to performance from differences related to ageing per se. To
circumvent this difficulty, we first took advantage of the fact that
for certain versions of the RAM task aged mice, like hippocampus-
lesioned mice, were able to express the same level of response
European Journal of Neuroscience, Vol. 17, pp. 628–640, 2003 � Federation of European Neuroscience Societies
doi:10.1046/j.1460-9568.2003.02464.x
Correspondence: Dr A. Marighetto, as above.
E-mail: a.marighetto@neurocog.u-bordeaux.fr�Present address: Columbia University Centre for Neurobiology and Behaviour, 1051
Riverside Drive, New York, NY10032, USA
Received 28 June 2002, revised 28 October 2002, accepted 19 November 2002
accuracy as adult mice. Previous neuroimaging studies (Bontempi
et al., 1999) have shown that, in adult mice, hippocampal metabolism
was increased significantly by RAM testing on a task that was very
similar to our simultaneous six-choice version. Therefore we hypothe-
sized that age-related differences should be observed in the hippo-
campal response to RAM testing even though the task used was this
(‘ageing- and hippocampal lesion- insensitive’) simultaneous six-
choice discrimination version. If so, one should also be able to show
that changing specific testing parameters would alter (e.g. reduce) the
age-related differences in brain activation. Specifically, we reasoned
that if, as expected, memory testing revealed stronger hippocampal
activation in adults with respect to aged mice, such a difference should
be suppressed (or at least reduced) by lengthening the retention
interval up to several weeks. Indeed, it was shown in the study of
Bontempi et al., in adult mice, that increasing the retention interval
from 5 to 25 days resulted in a reduction of retention testing-induced
activation of hippocampal metabolic activity. In other words, in
addition to equating performance among the two groups of aged,
lengthening the retention interval was used here as a mean to assess the
specificity of the putative age-related differences in brain activities
associated with memory testing.
Materials and methods
The cognitive impairment of senescent mice was shown by training the
animals in a three-pair discrimination task in the RAM (i.e. two-choice
discriminations, stage 1). The same mice were subsequently submitted
to a test-session (stage 2) where the manner of presenting the familiar
arms was changed to a simultaneous presentation of the six arms
altogether (six-choice discrimination). Such testing was aimed at
showing that aged mice did acquire significant knowledge about the
relative valence of the arms during stage 1 training although such
acquired knowledge could only be expressed when the presentation of
the arms was modified. The groups of animals submitted to RAM
training were killed for Fos immunochemistry following a retention
session of the six-choice discrimination performed after a period of
training remission (6 days) for half the animals and 30 days long for the
remainder. This second group corresponded to the first comparison
condition. The second comparison condition consisted in repeated
treadmill (TM) training performed in the same environment as RAM
training.
Animals
Mice were naive males of the C57/bL6 Jico inbred strain obtained from
IFFA Credo (Lyon, France). They were either aged mice of 21–
23 months old (n¼ 24) or adult mice of 4–5 months old (n¼ 24).
Upon arrival they were caged in groups, housed in a climatized
vivarium under a 12 : 12 h light : darkness cycle, and maintained with
access to food and water ad libitum. After 4–6 weeks, the animals were
caged singly a fortnight before the introduction of food deprivation,
which was introduced progressively over a number of days with the
animals eventually receiving a fixed amount of laboratory food chow
per day. Their body weight was maintained at a stable level, at
approximately 90% of their free feeding weight.
Mice of both age groups were allocated randomly into three
between-subject conditions (n¼ 8) according to three behavioural
regimes: (i) 6-day RAM; (ii) 30-day RAM; and (iii), 6-day TM. Mice
in conditions (i) and (ii) were both trained on a spatial discrimination
task in the RAM, but the two conditions differed with respect to the
retention interval, which was either 6 or 30 days before the last session.
Similarly, mice in the 6-day TM condition were given daily training on
the treadmill except for an interruption of 6 days before the last day of
training. Upon completion of the last day of training, all mice were
killed for immunohistochemistry. Eight mice were rejected from the
final analyses: four died before the end of the experiment and four
others yielded poor immunostaining because of inadequate perfusion.
The final number of aged mice in the three conditions (6-day RAM,
30-day RAM and 6-day TM) were 8, 6, and 5, respectively. The final
number of adult mice in the three conditions was seven for each.
All experimental manipulations were carried out in accordance with
the European Communities Council Directive of the 24 November
1986 (86/609/EEC).
Apparatus
Radial arm maze
This was a fully automated 8-arm RAM located in a quiet testing room
enriched with distal spatial cues; the dimensions and construction have
been described in detail elsewhere (see Marighetto et al., 1993). A door
was mounted at the entrance to each arm from the central platform and
a pellet dispenser was installed at the end of each arm. Door move-
ments were controlled by a computer program that also tracked the
position of the mouse within the maze continuously via pressure
detectors underneath the central platform and two pairs of photocell
beams installed along each arm. One pair of photocell beams guarded
the entrance of the arm, and another was placed just in front of the food
well. This enabled a real-time control of the accessibility to the arms
according to a predetermined test schedule.
Treadmill
This consisted of a Plexiglas enclosure measuring 30 cm long, 5 cm
wide and 20 cm high. A moving belt powered by a motor was mounted
on the floor of the enclosure.
Radial arm maze training
Shaping
Before discrimination training, the animals were habituated to the
apparatus over a period of 2 days. On each day, the mice were allowed
to move freely individually in the maze until all the food pellets
prebaited in the food well of each of the eight arms were collected.
Discrimination task
Each animal was separately assigned six adjacent arms of which three
served as positive (baited) arms and the remaining three served as
negative (unbaited) arms. The relative locations of these arms were
such that these six arms could be grouped into three pairs of arms with
opposing valence (designated as pairs A, B and C, as illustrated in
Fig. 1). In addition, it was ensured that if the positive arm of pairs A
and B was on the left (or right), then the positive arm of pair C had to be
on the right (or left).
The discrimination task comprised two consecutive stages, which
differed only in terms of the manner in which the arms were presented
to the mice. In Stage 1, only two arms (pairs A, B, or C) were acce-
ssible at any one time (i.e. two-choice discrimination), whereas all six
arms were accessible to the mice during Stage 2 (i.e. six-choice dis-
crimination). Such a design was based on our previous experiments
showing that most of the aged mice were impaired in acquiring the
three pairs (i.e. two-choice discriminations) as they failed to consis-
tently choose the positive arm within each pair. Nevertheless, after 15
sessions of such training, the same aged mice exhibited a preference
for positive arms similar to the one seen in their younger controls, given
that the pair presentation was changed to the simultaneous presentation
of the six arms altogether (i.e. six-choice discrimination).
Fos imaging of cognitive ageing in mice 629
� 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 17, 628–640
By equating the length of training at Stage 1 (i.e. 15 days), we also
needed to cater for a possible overtraining effect from developing in
subjects that might have acquired the task before the 15th daily session.
To this end, mice that had attained a predetermined criterion of
performance (on pairs A, B and C) before the 15th daily session were
trained on a second set of three pairs until the completion of the 15-day
period. The reward valence of the six arms was preserved since one
pair (pair [AB]) was formed by combining an arm from pair A with an
arm (of opposing valence) from pair B, and one pair was unchanged
(i.e. pair C). Lastly, a novel pair (pair N) was introduced which was
formed by the two arms that did not feature in pairs A, B or C. This
supplementary procedure was adopted in order to minimize the risk of
a possible over-training effect developing for the original three pairs
(pairs A, B and C). Performance on the variant task is not reported here
(see Marighetto et al., 1999 for a detailed description of performance
of aged and adult mice on such trials).
Stage 1: Concurrent two-choice discriminations. In each trial, the
subject had access to two adjacent arms with opposing valence (either
of pairs A, B and C). A choice was considered to be made when the
subject had reached the food well of an arm; this also triggered the
closure of the door to the alternative arm. The trial was finished as soon
as the subject returned to the central platform. The subject was then
confined to the central platform for 10 s before the next discrimination
trial began: this constituted the intertrial interval. Each daily session
consisted of 20 consecutive trials comprising alternate presentations of
pairs A, B and C according to a pseudo-random sequence. The mice
were trained on this task for a minimum of six sessions and a maximum
of 15 sessions. Within this interval, if they reached a predetermined
criterion of choice accuracy (see below), then training on the second
set of three pairs followed on the next day till the completion of 15
sessions. As explained above such a procedure was adopted to match
the amount of stage 1 training-sessions (i.e. 15 sessions of two-choice
discriminations) between the subjects and, at the same time, avoid
overtraining on the first set of pairs (A, B and C) in quick learners.
Choice accuracy was measured by the percentage of choice of the
positive arm (percent correct). A mouse was considered to reach crite-
rion performance when its overall choice accuracy was at or above
75% over two consecutive sessions, given that performance in each of
the three discrimination problems was at least 66% correct.
Stage 2: Six-choice discriminations. All subjects were transferred to
this stage on the 16th session of training. All the six arms composing
pairs A, B and C were used again here, and the reward contingency
among them remained unchanged but their presentation was modified.
At the beginning of each trial, the doors of the six arms were opened.
An entry into an arm was considered to be made when the subject
reached the food well of the particular arm. As soon as a subject was
back on the central platform, the door of the visited arm was closed but
those of the unexplored arms remained opened. The trial was ended as
soon as each of the three positive arms had been visited. This also
triggered the closure of the doors (of unexplored arms) when the mouse
has returned to the central platform. The mouse was confined there for
10 s before the next trial began. The session comprised five trials.
Accuracy was measured by the percentage of positive arms visited
among the first three visits of each trial.
Retention: six-choice discriminations. Following an interruption of
training of either 6 or 30 days for the 6-day RAM and 30-day RAM
groups, respectively, the mice were submitted to one more session of
six-choice discrimination training. This was exactly similar to the test
session as previously described.
Each mouse was killed 90 min after the beginning of this last train-
ing session.
Treadmill training
Treadmill control animals were paired with 6-day RAM mice and were
submitted to 16 daily sessions of TM training (5 cm/s). The duration of
each session was rendered as close as possible to the mean time spent
in the maze by RAM subjects (30 min on day 1, 20 min on days 2, 3 and
4, and 10 min per day from day 5 to the last day of testing). At the end
of each session, TM animals were given the same amount of food
pellets as that consumed by the RAM groups. After a 6-day rest
interval without training, animals were submitted to another 10-min
session of TM training, 90 min before being killed for immunohis-
tochemistry.
Fos-like immunohistochemistry and cell counting
Ninety minutes after the retention session, mice were deeply anaes-
thetized with sodium thiopental (70 mg/kg i.p.) and perfused through
the ascending aorta within 10 min with 100 mL saline solution (0.9%)
FIG. 1. General design of the experiment.
� 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 17, 628–640
630 K. Touzani et al.
followed by 200 mL of fixative containing 4% paraformaldehyde and
0.2% picric acid in 0.1 M phosphate buffer (pH 7.4). Brains were
removed and immersed in the same fixative for 2 h at room temperature
and then soaked overnight in a 20% sucrose solution in 0.1 M phos-
phate buffer, pH 7.4, at 4 8C. The brains were then frozen in cooled
methyl-2-butane and frontally sectioned in a freezing microtome at
50mm. One of each two consecutive sections was stained with thionine
and the second was treated for immunodetection of c-Fos protein. The
sections were collected in Veronal buffer pH 7.4 and rinsed twice with
the same buffer before immersion in a rabbit affinity purified poly-
clonal antibody (Oncogene Research Products, Boston, USA) raised
against amino acid residues 4–17 of human c-Fos protein. Sections
were incubated with the primary antibody at room temperature at a
1 : 2000 dilution in Veronal buffer and 0.4% Triton X-100 under
constant agitation. Approximately 24 h after incubation, sections were
rinsed with Veronal buffer and incubated with a biotinylated goat anti-
rabbit IgG (Jackson Immunoresearch Laboratories, PA, USA) 1 : 2000
in Veronal buffer, 0.4% Triton X-100 for 2 h. Sections were then
washed with Veronal buffer and incubated for 60 min with peroxidase-
conjugated streptavidin (Jackson Immunoresearch) 1 : 2000 in Veronal
buffer, 0.4% Triton X-100. The peroxidase activity was revealed
according to the glucose oxidase-nickel-DAB method (Shu et al.,
1988) and the development of reaction product was monitored under a
light microscope. Sections were then placed in sodium acetate buffer,
pH 6 for 5 min to stop the reaction, rinsed several times in distilled
water, mounted on gelatin-coated glass slides, dried overnight, rapidly
dehydrated through a graded series of ethanol, cleared in toluene and
then coverslipped with Eukitt mounting medium. Neurons with nuclei
exhibiting Fos-like immunoreactivity (FLI) were counted unilaterally
in sections taken through the dorsal hippocampus [CA1, CA3 and
dentate gyrus (DG)], the lateral septum (LS), the medial septum (MS)
and the medial prefrontal cortex (mPFC). For each area, the counts
were performed in three sections per mouse in a blind fashion. Cells
exhibiting FLI in the hippocampus and the septum were counted
unilaterally in the entire structure by caption of these brain regions
through a video camera connected to a light microscope. In mPFC,
Fos-immunoreactive nuclei were counted by direct microscopic obser-
vation within a 200mm2 grid per section viewed at 100�magnification
and positioned on the prelimbic subdivision according to the mouse
brain atlas of Franklin & Paxinos (1997).
Statistical analysis
Data were analysed by analyses of variance (ANOVAs). Each ANOVA
always included a between-subject factor, Age, which contrasted the
4–5-month and 21–23-month conditions. Analysis of behavioural data
also included a repeated-measure factor. Analysis of Fos data consisted
in comparisons between: (i) 6-day RAM and TM groups; and (ii), 6-day
RAM and 30-day RAM groups. This analysis included two between-
subject factors (Age and Behavioural condition) as well as a within-
subject factor (Region). Post hoc comparisons and correlation analyses
were performed using Scheffe’s pairwise comparisons and Pearson’s
test, respectively. An a level of 0.05 was required throughout.
Results
Behaviour
Stage 1: concurrent two-choice discriminations
All 14 adult mice acquired the three-pair discrimination task (mean
number of sessions to criterion 10.41� 0.71; minimum 6, maximum
15) but only 23.2% (3 out of 14) of aged mice managed to reach the
criterion performance within less than 15 sessions (respectively 7, 12
and 15 sessions to criterion for those three aged mice; mean number
11.33). Choice accuracy over the first and the last six sessions of this
stage for the adult and aged groups (comprising 6-day- and 30-day
RAM mice) is illustrated in Fig. 2. The performance of adult mice
improved over the course of training and approximated 80% correct in
the last session. Conversely, aged mice maintained a performance that
did not consistently vary from chance level over repeated training. The
performance was submitted to a 2� 6 ANOVA with the between-subject
factor Age and the within-subject factor Session. Within the first six
sessions, the analysis revealed a significant effect of Age (F1,26 ¼ 6.46,
P¼ 0.017) and its interaction with Session (F5,130¼ 4.68, P¼ 0.0006).
Within the last six sessions, there was a significant effect of Age
(F1,26¼ 33.5, P¼ 0.0001) and Session (F5,130¼ 5.6, P¼ 0.0001) as
well as their interaction (F5,130¼ 2.53, P¼ 0.032). These results
supported the conclusion that aged mice were significantly impaired
in acquiring the three-pair discrimination task.
Stage 2: six-choice discriminations
The progression of performance from the last session of training on
pairs A, B and C to the six-choice test is depicted in Fig. 3 (in the
aged group there were two missing values; data from two mice were
lost because of a computer failure). In the adult group, it can be seen
FIG. 2. Mean (�SEM) percentage of correct responses (choice of the positivearm) in the adult (6-day RAMþ 30-day RAM) and aged (6-day RAMþ30-day RAM) groups over the first and last six sessions before the attainmentof criterion performance in stage 1 (concurrent two-choice discrimination).Each mouse was trained until it reached the predetermined criterion ofacquisition on the first set of three pairs (A, B and C). Therefore the amount oftraining sessions required varied between individuals from a minimum of sixto a maximum of 15. This is the reason why the mean performance for eachgroup are presented for the first six and last six sessions of training. P< 0.05;P< 0.01; P< 0.001, vs. chance.
� 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 17, 628–640
Fos imaging of cognitive ageing in mice 631
that choice accuracy was decreased slightly in the six-choice session as
compared with the last session of three-pair discriminations, whereas
an opposite tendency was observed in the aged group.
A two-way ANOVA performed on these data with the between-sub-
ject factor Age and the within-subject factor Session (last-Stage 1 vs.
Stage 2) revealed significant effects of Age (F1,24¼ 29.49, P¼ 0.0001)
and its interaction with Session (F1,24¼ 6.8, P¼ 0.015). A simple
main effect analysis showed that there was a significant between-group
difference in the last session of Stage 1 (P¼ 0.001) but not in Stage 2
(P> 0.1). The disappearance of the group difference was mainly
because of a near significant tendency for improved response accuracy
in the aged group (repeated measures: P¼ 0.067) associated with an
opposite, although nonsignificant, tendency for an impairment in the
adult group (P> 0.10). This supports the conclusion that aged mice
were impaired in the two-choice but not six-choice version involving
the same set of items.
Retention of six-choice discriminations
This session followed an intermission of training of either 6 days (6-
day RAM groups) or 30 days (30-day RAM groups). Although 30-day
performance was slightly reduced in comparison with the 6-day
condition in both adult and aged groups (see Fig. 3), the effect of
retention-interval length was far from statistical significance.
A two-way ANOVA was performed on these data with both Age
and Interval (6-day RAM vs. 30-day RAM) as the between groups
factors and Session (last Stage 2 vs. Retention sessions) as within-
subjects factor. Neither the effect of Age (P> 0.1), nor the Inter-
val� Session (F< 1) and Age� Interval–Session interactions (F< 1)
were significant.
Immunohistochemistry
The mean numbers of Fos-positive nuclei in selected brain regions of
the septo-hippocampal system and in mPFC are depicted in Fig. 4, for
each group of mice, i.e. 6-day RAM experimental groups and control
groups (TM and 30-day RAM). Representative photomicrographs of
coronal sections from the adult and aged groups showing Fos-positive
nuclei in the different brain regions are presented in Fig. 5 (6-day
RAM), Fig. 6 (30-day RAM) and Fig. 7 (TM).
Comparisons between RAM and TM
Results summarized in Fig. 4 showed that, in adult mice, the number
of Fos-positive nuclei was greater in the 6-day RAM group than in the
TM group (training) in all the septo-hippocampal and mPFC regions.
In aged mice this RAM vs. TM between-group difference was nil in the
mPFC and was reduced in all the septo-hippocampal areas except for
DG. A three-way ANOVA with Age and Behaviour (RAM vs. TM) as
between-groups factors and Region (6 levels: CA1, CA3, DG, LS, MS
and mPFC) as the within-subjects factor performed on these data
confirmed this description, indicating a highly significant threeway
Age�Behaviour–Region interaction (F5,115¼ 9.11; P< 0.001). This
was mainly because of the absence of Age (F< 1) and of
Age�Region (F< 1) differences, whereas both the effect of Age
(F1,13¼ 10.05; P¼ 0.007) and its interaction with Region
(F5,65¼ 65.86; P< 0.001) were highly significant in RAM (6-day)
animals. These results support the conclusion that ageing affects the
Fos expression induced by RAM but not TM training in the mPFC and
in certain regions of the septo-hippocampal system.
There were also significant effects of Behaviour (F1,25¼ 17.34;
P< 0.001), Age (F1,25¼ 7.05; P¼ 0.014)) and their interaction (F1,25¼7.84; P¼ 0.01), indicating that RAM training globally increased
FLI contents as compared to TM, but that this increase was signifi-
cantly attenuated in aged mice. The effect of Region (F5,115¼ 97.1;
P< 0.001) and its interaction with Behaviour (F5,115¼ 15.69;
P< 0.001) and Age (F5,115¼ 10.61; P¼ 0.002) were also significant,
indicating thereby that the absolute level of Fos production, as well as
its differential between RAM and TM training and between ages varied
among the brain regions under investigation.
Additional Pearson analyses of correlation were performed on
FLI contents measured in each region within each group. Results
are summarized in Table 1. In adult mice, almost all FLI contents
were positively inter-related in the RAM (6-day) group but not
in the TM group. In contrast, no significant correlation was obser-
ved in aged mice of either the RAM (6-day) group or the TM
group. Although these analyses must be considered cautiously given
the small number of subjects in each group, they suggest that, in
addition to reducing Fos production associated with RAM training,
FIG. 3. (Left) Comparisons between the performance (mean�SEM percentage of correct responses) of aged (6-day RAMþ 30-day RAM) and adult (6-dayRAMþ 30-day RAM) mice in the last session of stage 1 and in the testing session of stage 2. (Right) Comparisons between the six-choice discriminationperformance of aged and adult mice in the testing session of stage 2 and in the retention session performed either 6 days (6-day RAM groups) or 30 days (30-dayRAM groups) later. ���P< 0.001 vs. aged; 888P< 0.001 vs. chance; 88P< 0.01 vs. chance; 8P< 0.05 vs. chance.
� 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 17, 628–640
632 K. Touzani et al.
ageing also affects the coherence of this production across brain
regions.
Finally, correlation analysis revealed positive interrelations between
FLI contents in several regions and RAM performance in adult mice
(LS r¼ 0.84, P¼ 0.02; MS r¼ 0.73, P¼ 0.06; PFC r¼ 0.78, P¼ 0.04;
for CA1, CA3 and DG; all P> 0.14) but not in aged mice (all r< 0.13
and all P> 0.18).
Brain activation in the subgroup of aged mice displaying
no sign of impairment in stage 1
As detailed in the method section, stage 1 training in the two-choice
discrimination design was not strictly identical among the subjects.
This comes directly from our twofold aim to match the amount of
training across subjects and to avoid possible over-training effects in
fast learners. Thus, all mice reaching a predetermined criterion of
performance on the first set of three pairs before the 15th session (i.e.
all adult mice and three aged mice) were submitted to training on a
second set of three pairs, whereas the mice that failed to acquire the
two-choice task (i.e. all aged mice but three) were trained on the same
set of pairs till the fifteenth session. As can be seen in Fig. 4, the
subgroup of aged mice that displayed no sign of deficit in stage 1 (and
therefore underwent the exact same training as the adult group)
nevertheless displayed patterns of RAM-induced Fos production that
were indistinguishable from that seen in the whole set of aged subjects.
FIG. 4. Mean (�SEM) number of Fos-positive nuclei in different brain regions in each group following testing in the six-choice discrimination. The ‘unimpairedaged group’ was made of those three aged mice, which reached the criterion performance in the concurrent two-choice discrimination task (pairs A, B and C)within less than 15 sessions. Therefore, exactly as the younger adult mice, these three aged mice underwent training on the second set of three pairs ([AB], C & N)till the 15th session of two-choice discrimination training. 8P< 0.05; 88P< 0.01 vs. aged; �P< 0.05; ��P< 0.01 vs. 30-day RAM; þP< 0.05; þþP< 0.01;þþþP< 0.001 vs. 6-day RAM.
� 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 17, 628–640
Fos imaging of cognitive ageing in mice 633
Therefore, this observation enables us to exclude the slight between-
subjects differences in the way animals completed initial training
with pairs as a potential cause for the age-related differences in
Fos production observed following testing in the six-choice version
of the task.
Comparison with the long-term retention condition
As can be seen in Fig. 4, FLI contents measured in the septal area
and in the hippocampal CA1 and CA3 subfields of the adult RAM
group of mice were reduced following the long (30-day) with respect to
the short (6-day) retention intervals. Conversely, these contents were
very similar among the two retention intervals in both the DG and
mPFC.
In the aged subjects, the effect of the retention interval on FLI
contents was almost nil in both the septal area and in the hippocampal
CA1 subfield, whereas, as observed in adult mice, increasing the
retention interval was associated with a reduction of Fos production
in CA3. Surprisingly, the content of FLI measured in the mPFC in this
aged group was significantly greater for the long- than for the short
retention interval.
These impressions were confirmed by a three-way ANOVA with
Age and Interval as between-groups factors and Region as the
within-subjects factor indicating a significant Age� Interval–Region
triple interaction (F5,120¼ 5.47; P< 0.001). This was mainly because
of the lack of effect of Age (F< 1) and of its interaction with Region
(F< 1) in the 30-day RAM groups, a result in sharp contrast with
observations previously described in the 6-day RAM groups (see
above), wherein both effects were highly significant. This was also
reflected by a significant Age–Interval interaction (F1,26¼ 4.48;
P¼ 0.045). Finally, the analysis also revealed a significant effect
of Region (F5,120¼ 104.74; P< 0.001) and of its interactions with
both Age (F5,120¼ 8.73; P< 0.001) and Interval (F5,120¼ 2.57;
P¼ 0.03), suggesting that the absolute level of FLI, as well as its
differential between adult and aged groups, and between short and long
retention intervals, varied among the anatomical structures investi-
gated.
These data support the conclusion that in the mPFC and certain
regions of the septo-hippocampal system ageing selectively reduces
Fos expression associated with short-term but not long-term retention
of the RAM discrimination.
FIG. 5. Photomicrographs of coronal sections from young (left column) and aged (right column) groups showing Fos-positive nuclei in the hippocampus (A and D),lateral septum (B and E) and medial prefrontal cortex (C and F) in the 6-day RAM groups. Scale bar, 0.4 mm.
� 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 17, 628–640
634 K. Touzani et al.
Additionally (see in Table 1), Pearson correlation analysis showed
that FLI contents measured in regions of the septo-hippocampal
system in the adult 30-day RAM group were again positively inter-
related. However, there was no correlation between regions of the
septo-hippocampal system and mPFC as previously observed in the 6-
day condition. Furthermore, in aged animals, a pattern of interrelations
similar to that seen in adults was observed in the 30-day RAM group,
whereas no such inter-relations were actually visible in the 6-day
condition. This suggests that ageing alters the coherence of brain
responses for the short- but not the long-term retention of the dis-
crimination task.
Finally, in the adults, there was no significant relationships between
FLI contents in septo-hippocampal and mPFC regions and RAM per-
formance at the 30-day retention interval (all r< 0.24 and all P> 0.14).
In the aged mice, there was a tendency towards inverse relations
between performance and FLI contents in regions of the septo-hippo-
campal system, although most of them were far from statistical
significance (vs. CA1 r¼�0.73, P¼ 0.09; vs. CA3 r¼�0.72,
P¼ 0.11; vs. DG r¼�0.85, P¼ 0.03; vs. LS r¼�0.65, P¼ 0.17).
Discussion
The behavioural data showed that the performance of aged mice was
significantly diminished in comparison with that of the adult groups in
stage 1 discriminations, but not in the stage 2 testing involving the
same set of RAM arms. Thus, despite prolonged training, aged mice
failed to demonstrate a consistent preference for the positive arms in
the two-choice situation, whereas such a preference was actually
expressed in over-chance performance as early as the first session
of six-choice discriminations. The present study therefore confirms our
previous findings in a novel manner by showing contrasting effects of
ageing between the different versions of this position discrimination
task. This notion is discussed in the next section.
Although the different groups of RAM mice performed the six-
choice version of the task at about the same level of accuracy at the end
of the experiment, immediately before they were killed, Fos imaging
revealed that the brain sites investigated were not recruited similarly
within the different conditions of age and retention-interval length.
The significance of such differences in brain response to RAM training
FIG. 6. Photomicrographs of coronal sections from young (left column) and aged (right column) groups showing Fos-positive nuclei in the hippocampus (A and D),lateral septum (B and E) and medial prefrontal cortex (C and F) in the 30-day RAM group. Scale bar, 0.4 mm.
� 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 17, 628–640
Fos imaging of cognitive ageing in mice 635
will be discussed subsequent to the behavioural section with respect to
both the effects of the retention interval in the adults and to the ageing
effect.
Selectivity of senescence-related memory impairment
Experiments using variants of the original RAM design (Marighetto
et al., 1999, 2000; Etchamendy et al., 2001) have shown that aged mice
as well as hippocampal-lesioned mice (Etchamendy et al. in press)
display a bias towards positive arms similar to that seen in younger
adult controls, whenever the arms are made accessible to them
successively one by one (in a go-no-go procedure) or all at a time
(in a multiple choice procedure as the one used in the present
experiment). Conversely, whenever the same aged and lesioned mice
are offered a choice between only two of the same adjacent arms,
they significantly differ from their younger controls in that they fail
to choose the positive arm more often than the negative one. Given
that the basic requirements of the task are mainly identical between
the different testing conditions, such a selective age-related deficit
might be viewed as cognitive in nature. Furthermore, it reveals that
different forms of mnemonic expression for the same piece of
past experience can be assessed in the same subjects through a change
in arm presentation. On the basis of the theorization proposed by
Eichenbaum (1992) and Eichenbaum et al. (1994), we speculate that
the two-choice version would require the ability to mentally compare
and contrast the representations associated with each arm in order to
inform an explicit relative judgement between the available choices.
Therefore, two-choice discrimination would critically rely on a com-
plex kind of mnemonic representation, incorporating the relative
relationships among choice arms, which normally enable comparisons
and contrasts between separately experienced items. By contrast, in
versions that are less demanding with regard to comparison processing
(e.g. when only one arm is opened or too many arms are accessible at
any time), position discrimination could also rely on an adapted
response to each arm which in turn is based on stimulus-response
habit strengths (McDonald & White, 1993; Packard & McGaugh,
1996). Within this view, the deficit seen in aged mice is qualitatively
similar to the preferential loss in declarative/explicit memory seen in
human senescence (Craik & Jennings, 1992; Schugens et al., 1997).
FIG. 7. Photomicrographs of coronal sections from young (left column) and aged (right column) groups showing Fos-positive nuclei in the hippocampus (A and D),lateral septum (B and E) and medial prefrontal cortex (C and F) in the 6-day TM group. Scale bar, 0.4 mm.
� 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 17, 628–640
636 K. Touzani et al.
The present Fos imaging study gives further support to our view in
showing that the hippocampus is likely to contribute to the mediation
of adult performance and disturbances in such hippocampal mediation
occur with ageing. However, the present data can also be viewed as
contradictory with our hypothesis as the pattern of testing-induced
hippocampal activation was similarly altered with respect to the adult
group, in the subgroup of aged mice that showed no sign of an
impairment in learning the three-pairs discrimination task. This latter
aspect of our study will be discussed first.
Consistency of the senescence-related alteration in testing-induced Fos production
As in our previous study (Marighetto et al., 1999), we observed here
that about one third of the aged group could acquire the two-choice
discrimination task in stage 1 as quickly and accurately as the adult
control mice. Nevertheless, even those ‘apparently unimpaired’ aged
mice subsequently failed in recombination trials where relational
memory was assessed specifically (% correct was 45 in the aged
vs. 72 in the adult group). As previously discussed in detail, spared
initial learning of two-choice discriminations in ‘unimpaired’ aged
mice is likely to rely on a different cognitive strategy than in the adults.
One such a strategy would restrain the need for explicit between-arms
comparisons supposedly required in that task thereby supplying to
their (subsequently revealed) relational memory deficit. Whatever the
case, the reliability of the deficit evidenced in our paradigm in the aged
population might explain the present similarity in hippocampal Fos
expression between ‘unimpaired’ and impaired aged mice. Therefore,
not only are the present Fos data with our hypothesis that senescence is
accompanied by significant alteration in hippocampal-dependent rela-
tional processes, but they also support our previous assumption that
such a dysfunction might be less variable among individuals than
classically believed to be (see Marighetto et al., 1999 for further
discussion).
The consistency of patterns of brain activation among the aged
population also enables us to rule out a methodological concern related
to existing between-subjects differences in the completion of stage 1
training. Indeed, the subgroup of ‘unimpaired’ aged mice underwent
the exact same RAM training as the adult group (i.e. training on a
second set of pairs after acquiring the first three pairs), and never-
theless displayed a significant reduction in hippocampal activation.
Therefore, qualitative differences in stage 1 training might not be
considered as the critical factor for explaining the observed between-
age differences in testing-induced Fos production.
Effect of retention-interval length or age onpattern of brain activation
The present immunohistochemical data from the adult groups show
that the short-term retention of the six-choice discrimination task
produces a significant activation of the septo-hippocampal system,
in comparison with locomotor activity training. This observation is in
line with a report on the Fos imaging in different RAM memory tasks
in rats (Vann et al., 2000). Within the hippocampus, the greatest over-
expressions induced by the RAM testing were observed in CA1 and
CA3 subfields (CA1¼CA3>DG). These subfield-specific changes
extend the findings of Hess et al. (1995) who showed that rats
performing a well-learned odour discrimination task had increased
c-fos mRNA expression in the hippocampus, with the greatest effect
being found in CA1. Frontal FLI contents were also increased in our
RAM-trained adult mice. Furthermore, positive inter-relations
between the different regions investigated were observed, although
they were not visible in TM controls. This could indicate the simulta-
neous recruitment of the septo-hippocampal and frontal areas as parts
of the same extended network mediating the short-term retention of the
RAM six-choice discrimination task.
The RAM testing-induced activation in adult brains was reduced in
all sites investigated, except DG and mPFC, when the retention
interval was increased from 6 to 30 days. In short-term retention,
Fos expression induced by testing was also markedly reduced in aged
brains with respect to adult ones, in all sites except the DG. Both these
retention interval- and age-dependent patterns of brain activation were
TABLE 1. Correlation matrix tables for Fos-like immunoreactivity in brain regions for each experimental group
Adult Aged
CA1 CA3 DG LS MS CA1 CA3 DG LS MS
6-day RAMCA1CA3 0.797� 0.525DG 0.933�� 0.793� 0236 0.699LS 0.802� 0.759� 0.843� 0696 0578 0.565MS 0713 0.67 0.68 0721 �0177 0246 0008 0.228mPFC 0.802� 0.839� 0718 0.867� 0.868� 0531 0.948�� 0533 0358 0.084
30-day RAMCA1CA3 0.878�� 0.789DG 0.821� 0.982��� 0.877� 0.807�
LS 0.873� 0.859� 0.759� 0.82� 0.811� 0.947��
MS 0667 0495 0.33 0.841� 0085 �0116 0313 0.362mPFC �0001 �0225 �0313 0191 0343 0251 0027 0359 0.55 0.78
6-day TMCA1CA3 �0.294DG 0633 �0494 0.783LS 0554 0357 0679 �0287 �0.097MS �0246 �0423 0.42 0802 0157 0117 0.767mPFC �0277 �0443 0484 0713 0.975�� �0293 �0381 0.82 0.867
�P< 0.05, ��P< 0.01, ���P< 0.001, Pearson Correlation
� 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 17, 628–640
Fos imaging of cognitive ageing in mice 637
observed despite the lack of between-groups difference in the accuracy
in performing the six-choice discrimination task. Therefore, the basic
requirements of the task (in terms of reward, stimulus and response
involved) can be rejected as potential causes for the septo-hippocampal
activation induced by RAM testing at the short (6-day) retention
interval. However, the fact that apparently similar performances can
be sustained by a differential contribution of the same (here septal and
hippocampal) brain regions can be interpreted in at least three different
ways.
A first possible explanation is the absence of causal relationship
between septo-hippocampal activation and six-choice discriminative
performance. Even though such a hypothesis cannot definitely be
rejected, we will defend the position that activation of the hippocam-
pus has meaningful implications for discrimination performance. Our
opinion is based on recent data (Etchamendy et al., 2003) showing that
although hippocampus-lesioned mice performed the six-choice ver-
sion of the task with the same accuracy as their sham controls, both
groups displayed significant qualitative differences since discrimina-
tive behaviour appeared to be based on a cognitive strategy similar to
the one used in go-no-go discriminations in the former but not in the
later group. Supporting the view that six-choice discrimination might
rely on different kinds of cognitive processes mediated by dissociated
memory systems, these observations led us to favour a second possible
interpretation of our set data as follows.
Although quantitatively similar, the performances were qualita-
tively distinct between the two conditions of retention-interval or
age, i.e. relied on different memory systems within which the
septo-hippocampal region is not equally involved. Thus, we already
postulated that existing relational representations of past episodes in
adult control mice would sustain flexible expression of memory in all
various forms of between-arms discriminations. Meanwhile, as a result
of hippocampal dysfunction, position discrimination in aged mice
would mainly rely on stimulus-response habit. The present age-related
reduction of Fos production in CA1 and CA3 associated with short-
term RAM testing condition supports our view. If we now speculate
that, as time passes, the full richness of relational representation
progressively wanes, then this implies that the mnemonic representa-
tions of adults progressively become qualitatively similar to those of
the aged. Hence, after a long retention interval, discriminative
responses of adult mice might rely on simple associations, mainly
like those of aged mice in short-term retention, and the pattern of brain
responses to long-term retention testing would more closely corre-
spond to a procedural or implicit expression of memory. In this view,
first, the relative reduction in testing-induced hippocampal activation
in the long term condition is coherent with human neuroimaging data
showing that significant activation of the hippocampal formation is
specifically associated with conscious recollection of the learning
episode (Schacter et al., 1996; Eldridge et al., 2000). Second, the
absence of any age-related difference in brain responses to RAM
testing at the 30-day retention interval is coherent with the idea that
ageing is associated with a preferential alteration in the system that
mediates declarative memory.
A third alternative interpretation of our results could be that whether
in aged or in younger mice, in short- or in long-term retention, the six-
choice discrimination task always relies on the same cognitive pro-
cesses, these being possibly mediated by the recruitment of different
neural networks. In fact, such a ‘brain reorganization’ hypothesis is
frequently encountered (at present) in the current literature devoted to
cognitive ageing in humans (Hazlett et al., 1998; McIntosh et al.,
1999). Similarly, a gradual reorganization of the neural substrates
underlying long-term memory storage is postulated in a model of
memory consolidation proposed by Squire & Alvarez (1995). Accord-
ing to this model, the hippocampus is only temporarily involved in
the maintenance of acquired information until such a reorganiza-
tion (i.e. the consolidation process) has ended. Such a view might
indeed explain why the hippocampus was not activated significantly by
the long-term retention of the task in adult mice and, as a direct
consequence, why the differential between the two ages disappeared.
However, as explained below, such an interpretation can hardly account
for the total absence of ageing effect in the long-term condition.
Selectivity of senescence-related changes inthe pattern of brain activation
The present Fos imaging study reveals a triple selectivity of the effect
of ageing on brain responses associated with testing in the six-choice
discriminations task: (i) ageing affects responses to RAM but not TM
training; (ii), ageing reduces c-Fos expression in some but not all brain
regions as FLI contents measured in DG were identical in aged and
adult mice, whatever the condition. This age-related decrease in the
number of Fos-positive neurons within the hippocampus is unlikely to
result from neuronal death as Rapp & Gallagher (1996) clearly
demonstrated, by using modern stereological methods (which pro-
vided estimates of total neuron number in a given region in stead of
measures of neurons density), that hippocampal neuron number was
relatively preserved in aged rats; and (iii), finally, ageing alters Fos
responses in terms of both their amplitude and their inter-area relation-
ships, in the condition of short- but not long-term retention of the RAM
task. This latter selectivity is particularly remarkable and the possi-
bility that it was a pure consequence of the retention interval effect
observed in the adults can be excluded for the following reasons. First,
in mPFC, the relative increase in Fos products in the aged mice was nil
after the 6-day retention but strictly normal after the 30-day retention,
whereas in the adults this particular area was activated equally and
significantly in both conditions of RAM testing. Second, in the adult
groups, although the FLI contents observed in most of the septo-
hippocampal regions (areas) were diminished as a result of the increase
in the retention interval length, they were nevertheless inter-related
positively in both RAM conditions, whereas they were not in the TM
group. Conversely, in the aged mice, these Fos measures were mainly
unchanged in terms of their amplitudes between the 6-day RAM and
the 30-day RAM groups, but they were positively interrelated only in
the latter group. In summary, Fos imaging of septo-hippocampal and
frontal responses to the discrimination task indicates that ageing
selectively modifies the brain response to the short-term retention
of the task.
This selectivity enables us to rule out a serious methodological
concern that is intrinsically related to the use of an indirect correlate
of neural activity to image age-related changes in brain function
(D’Esposito, 1999). Namely, if ageing affects the ability of cells to
express the gene c-fos (or the kinetics of such expression) as well as
neural activity, then age-related differences in testing-induced Fos
activation could be misleading. The risk of such possible confusion is
clearly reduced whenever it can be demonstrated that age-related
differences in the activation of a particular brain area are not obser-
ved systematically but in response to certain kinds of stimulation.
Such is precisely the case here, where the mPFC in aged brains was
normally activated in response to long-term but not short-term reten-
tion testing.
Consequently, the present age-related reductions in certain Fos
protein expressions add to a previous report on the age-related increase
in hippocampal c-fos transcription induced by high-frequency stimu-
lation (Lanahan et al., 1997) to suggest possible modifications in the
activation of signalling pathways upstream of c-fos. Our study further
shows that the apparent coherence of the brain response, in particular
� 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 17, 628–640
638 K. Touzani et al.
within the septo-hippocampal areas, might or might not be normal in
senescent mice depending on the behavioural situation. Taken as a
whole, our Fos data are therefore strongly suggestive of age-related
disturbances in the connective processes that sustain proper recruit-
ment of a set of structures in some, but not all, circumstances. It is
noteworthy that human data relative to age-related changes in brain
imaging of cognition are also frequently interpreted in terms of
changes in effective connectivity in the neural network underlying
the task (Cabeza et al., 1997; Esposito et al., 1999; McIntosh et al.,
1999).
Conclusion
The present neurofunctional correlates of ageing support our hypoth-
esis that a relational/declarative form of memory undergoes prefer-
ential loss during ageing. Even though the whole set of our existing
data from ageing and lesion experiments is coherent with our inter-
pretation, the precise nature of the processes involved remains spec-
ulative. More experiments are clearly needed to interpret more closely
the seemingly complexity of senescence-related changes in brain
activity associated with cognitive performance in our mice. In any
event, the present study shows c-fos protein expression to be a valuable
tool for such neuroimaging approaches. Our study also demonstrates
that, as in humans, it is possible to evidence subtle and selective effects
of ageing on both the brain and behaviour in mice.
Acknowledgements
This work was supported by the C.N.R.S and the Conseil Regional d’Aquitaine.The authors are very grateful to Drs B. K.Yee, J. Micheau and J. L. Guillou fortheir helpful comments on previous versions of the manuscript.
Abbreviations
DG, dentate gyrus; FLI, Fos-like immunoreactivity; LS, lateral septum; mPFC,medial prefrontal cortex; MS, medial septum; RAM, radial arm maze; TM,treadmill.
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