Neurobiology of Learning and Memory 80 (2003) 32–41

www.elsevier.com/locate/ynlme

Involvement of the hippocampal CA3-region in acquisition and

in memory consolidation of spatial but not
in object information in mice
Gr
egory Stupien, C
edrick Florian, and Pascal Roullet*
Centre de Recherches Sur la Cognition Animale (CRCA), CNRS FRE 2382, Universit

e Paul Sabatier, 118 route de Narbonne, 31062
Toulouse Cedex 4, France
Received 30 May 2002; revised 14 February 2003; accepted 18 February 2003

Abstract

This study investigates the implication of the hippocampal CA3-region in the different phases of learning and memory in spatial
and non-spatial tasks. For that purpose, we performed focal injections of diethyldithiocarbamate (DDC) into the CA3-region of the
dorsal hippocampus. The DDC chelates most of the heavy metals in the brain which blocks selectively and reversibly the synapses
containing heavy metals, i.e., the mossy fibres synaptic buttons and synapses of the dendrites of pyramidal cells. The effects of
temporal inactivation of the CA3-region was examined in a non-associative task, the spatial open-field, designed to estimate the
ability of mice to react to spatial changes, and in the object recognition task, designed to estimate the ability of mice to identify a
familiar object. The results show that DDC induced a specific impairment on learning and memory consolidation in the spatial
open-field but had no effect on recall in this task. In the object recognition task, DDC did not induce any impairment in the different
phases of learning and memory. These data demonstrate that the hippocampal CA3-region is specifically implicated in spatial
information processing and seems to be involved not only in acquisition but also in consolidation of spatial information.
Ó 2003 Elsevier Science (USA). All rights reserved.

Keywords: Spatial learning; Spatial memory; Massed procedure; Hippocampus; CA3-region; Mice

1. Introduction
Memory is often separable in interdependent but
potentially dissociable processes including acquisition,
consolidation, and recall (Abel & Lattal, 2001;
McGaugh & Izquierdo, 2000; Riedel et al., 1999).
Studies on these different phases of memory are gener-
ally realised on associative tests as single-trial passive
avoidance task (Gold, 1986; Izquierdo et al., 1992; Iz-
quierdo & Medina, 1997) and recently on the fear-con-
ditioning task (Bourtchouladze et al., 1998; Nie & Abel,
2001). On the other hand, in complex learning (e.g.,
spatial learning), the majority of studies are conducted
without distinguishing these different phases of memory.
These studies have shown the role of different structures
in spatial learning such as the prefrontal cortex (Harri-
*
Corresponding author. Fax: +33-5-61556154.
E-mail address: roullet@cict.fr (P. Roullet).
son & Mair, 1996; Mogensen, Pedersen, Holm, & Bang,
1995; Sutherland, Kolb, & Whishaw, 1982), the nucleus
accumbens (Roullet, Sargolini, Oliverio, & Mele, 2001;
Usiello et al., 1998) and particularly the dorsal hippo-
campus. In fact, lesion of the dorsal part of the hippo-
campus decreases performances in associative spatial
tasks like the radial maze (Olton, Walker, & Gage, 1978;
Jarrard, 1983) or the Morris water maze (McDonald &
White, 1994; Morris, Garrud, Rawlins, & OÕKeefe,
1982). In a non-associative spatial task, the spatial open-
field, our previous studies have shown that prelimbic
cortex lesion and fimbria–fornix lesion induces a selec-
tive impairment in the ability of mice to react to the
spatial change (Sargolini, Roullet, Oliverio, & Mele,
1999). However, in this study and in all studies which
use irreversible lesions realised before the beginning of
tasks, it is impossible to know which phases of the
memory process lesions affect (Abel & Lattal, 2001). To
study the different phases of memory, it is necessary to

1074-7427/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved.
doi:10.1016/S1074-7427(03)00022-4
 G. Stupien et al. / Neurobiology of Learning and Memory 80 (2003) 32–41
block temporarily the hippocampus. This is generally
done with a short-acting drug injected at various times
before or after training (Abel & Lattal, 2001; McGaugh
& Izquierdo, 2000). Nevertheless, one can use also the
diethyldithiocarbamate (DDC), which chelates most of
the heavy metals (Danscher, Haug, & Fredens, 1973;
Holm, Andreasen, Danscher, & Nielsen, 1991; Szer-
dahelyi & Kasa, 1987). In the hippocampal slice, DDC
blocks the induction of mossy fiber LTP (Lu et al.,
2000). This chelator has been used before to block
temporarily the hippocampus (Frederickson, Freder-
ickson, & Danscher, 1990) and recently in our labora-
tory, to block the hippocampal mossy fibres (Lassalle,
Bataille, & Halley, 2000). The maximal duration action
of DDC into the dorsal hippocampal of the rat ranges
from 1 h (Lees, Cuajungco, & Leong, 1998) to 2 h (Holm
et al., 1991) and causes a time-dependent bleaching of
Timm staining pattern (Danscher et al., 1973; Freder-
ickson et al., 1990; P
erez-Clausell & Danscher, 1986;
Szerdahelyi & Kasa, 1987). With this short duration of
action shown in rats, the DDC allowed us to work on
the three phases of memory.
The dorsal hippocampus is a heterogeneous structure
that consists of a largely unidirectional loop of excitatory
pathways through different subfields, the dentate gyrus,
CA3, CA1, and subiculum (Jones & Smith, 1980). The
majority of the hippocampal studies focus on the CA1-
region and have shown a clear implication of this subfield
in spatial memory (Gilbert, Kesner, & Lee, 2001; Olsen,
Scheel-Kr€ uger, Moller, & Jensen, 1994; Volpe, Davis,
Towle, & Dunlap, 1992). The CA3-region seems also to
be implicated during spatial learning (Kesner, Gilbert, &
Wallenstein, 2000) and this subfield seems to be impor-
tant during the acquisition phase but not in the recall
(Kesner et al., 2000; Lassalle et al., 2000). However, to
our knowledge, nothing is known about the role of this
area in the phases of memory consolidation. This CA3-
region holds a strategic position in the hippocampus
because it receives sensorial information from the
external and internal environment via two principal
pathways, mossy fibres and the perforant path (Amaral
& Witter, 1989; Dolorfo & Amaral, 1998). In addition,
this structure is considered to be an auto association
network for storage and retrieval of information partic-
ularly in working memory processes (Rolls & Treves,
1998; Treves & Rolls, 1992).
The aim of this experiment is to investigate the
hippocampal CA3-regionÕs implication in the three
phases of spatial and non-spatial learning. We have
used DDC injections into the complete hippocampal
CA3-region to block synaptic transmission at the
mossy fibres level as well as into pyramidal cells. First,
since most experiments with DDC treatment have
been realised on the rat (Danscher et al., 1973; Fred-
erickson et al., 1990; Holm et al., 1991; Szerdahelyi &
Kasa, 1987), it was important to determine the exact
33
duration of DDC action in mice in order to know
when this treatment blocks the CA3-region during the
different phases of learning. Second, we have inacti-
vated hippocampal CA3-region during acquisition,
memory consolidation, and the recall process of spatial
information in the spatial open-field task. In this non-
associative task, mice have to memorise a spatial
objects configuration in less than 40 min, a task that
can be used to study the different phases of memory
(Roullet et al., 2001). It consists of placing mice in an
open-field containing five objects and, after three ses-
sions of habituation, examining their reactivity to ob-
ject displacement. Control animals show an increased
exploration of the displaced object. This response is
usually interpreted as an index of the ability of
animals to detect and react to the spatial change
(Poucet, 1989; Roullet, Mele, & Ammassari-Teule,
1996; Thinus-Blanc, Durup, & Poucet, 1992). This task
is realised within one learning session to avoid multi-
ple injections of DDC but also to allow working only
on the first information memory consolidation. This
point is very important because when learning sessions
are done during several days, memory trace is reacti-
vated and reconsolidated successively during each
learning session. Implicated structures or molecular
mechanisms can be different during first memory
consolidation or during reconsolidation after reacti-
vation of the memory trace (Nader, Schafe, & Le
Doux, 2000; Przybyslawski, Roullet, & Sara, 1999;
Sara, 2000). For this reason, it is very important to
use a one-day procedure. Third, we have blocked the
hippocampal CA3-region in the three different phases
of memory in the non-spatial task, the object
recognition task. This task uses differential level
exploration between familiar and unfamiliar objects as
a behavioural measure for recognition (Ennaceur &
Delacour, 1988). It is based on the animalÕs sponta-
neous preference for novelty. If the hippocampal CA3-
region were implicated only in spatial memory, we
would expect DDC injected mice to be severely
impaired in spatial open-field but to display normal
behaviour in the object recognition task.
2. Experiment 1: Duration of DDC action
The aim of this first experiment is to determine the
exact duration of maximal DDC efficacy. This infor-
mation is essential to devise an experimental behavio-
ural procedure for the spatial and non-spatial task in
acquisition experiment. In fact, is imperative that DDC
is effective during all learning sessions in all experiments.
In the memory consolidation experiments it is also
important to know whether DDC acts during the whole
duration of memory consolidation or if it acts only
during the first step of this phase.
34 G. Stupien et al. / Neurobiology of Learning and Memory 80 (2003) 32–41
2.1. Materials and methods
In the three experiments, subjects were males and
females C57BL/6 inbred mice born in our laboratory.
After birth, they remained with their two parents until
weaning at 21 days. Then they were placed in groups of
three to six animals of the same sex and same age, in
standard breeding cages (21
21
12 cm) placed in a
rearing room at a constant temperature (23  1 °C) with
food and water ad libitum.
Every possible effort was made to minimise animal
suffering and all procedures were in strict accordance
with European community and French national laws
and regulations on the use of animals in research and
NIH guidelines on animal care.
2.1.1. Surgery
At the time of surgery, mice were 90 to 120 days
old. They were anaesthetised with chloral hydrate
(400 mg/kg) and placed in a stereotaxic apparatus (Da-
vid Kopf Instruments) with mouse adapter and lateral
bars. The head skin was cut longitudinally and the in-
jection needle was introduced into the dorsal part of the
right hippocampus. The following coordinates with
lambda and bregma in the same horizontal plane were
used: posterior to bregma, )1.6 mm; lateral to midline,
2 mm; and )2.6 mm beneath skull surface (Franklin &
Paxinos, 1997).
2.1.2. Drug and Timm’s staining
Twenty mice received an unilateral stereotaxic injec-
tion of 0.25 ll sodium diethyldithiocarbamate (200 mM)
into the dorsal part of the right hippocampus. The un-
treated left side was considered as an internal control.
After well-defined delay (15 min to 5 h), animals are
perfused with a sodium sulphide solution according to
the Timm method (Timm, 1958a, 1958b).
2.1.3. Histology
Brains were removed, frozen at )20 °C and 40 lm
frontal microtom sections were obtained. Further pro-
cessing for the neo-Timm method was carried out as
described by Danscher (1981). To perform the sulphide
silver staining mounted sections were placed in jars and
covered with the physical developer. The staining itself
took place in the dark with the jars placed in a bath held
constantly at 26 °C.
2.1.4. Densitometric analysis
The densitometric analysis was performed on sections
from 20 animals with successful DDC injections. Sec-
tions were digitised using a Zeiss Axioplan microscope
surmounted by a JVC-F30 video camera. The pictures
collected (Fig. 1) were analysed with Photoshop soft-
ware. This software allowed to work with grey levels and
to determinate the percent of grey in the different parts
Fig. 1. TimmÕs staining and DDC injection. (A) Hippocampal for-
mation of a control mouse stained with the Timm sulphide-silver
method. The mossy fiber system of the CA4–CA3 region (mf) is in-
tensely stained, the basal (bd) and apical (ad) dendrites of the hippo-
campal subfields CA3 and CA1 exhibit moderate staining intensity,
while the striatum lacuno-moleculare (lm) and the granular part of
CA4-region are not colored. (B) Staining pattern of the hippocampus
in the DDC treated mouse (0.25 ll/side; 200 mM), 90 min after injec-
tion. We observe a bleaching of TimmÕs staining in the synaptic fields
of the hippocampus previously cited.
of hippocampus, i.e., mossy fibres and striatum lacuno-
moleculare. The striatum lacunosum and the striatum
moleculare were used as a staining reference to the in-
jected and non-injected sides (they are devoid of zinc
and present a low variation of density after TimmÕs
staining treatment). Among several baths, it is possible
to find differences in staining intensity. For this reason,
it was essential to determine the level of grey in the
mossy fibres for each section in relation to aspecific
background staining. The relative difference in staining
intensity between the injected and non-injected hippo-
campal regions was expressed by the index of bleaching:
 
mf B  lmB
Index ¼ 1 
100;
mf A  lmA
where the subscripts mf and lm represent the level of
grey in the mossy fibres and the striatum lacuno-mo-
leculare, respectively. The indices A and B refer to the
non-injected and injected sides.
2.2. Results
Injection of DDC into hippocampal CA3-region
prior perfusion with sulphide resulted in a localized
bleaching of the Timm staining pattern and the extent of
this bleaching varied with time (Fig. 2). Already 15 min
after the injection, DDC has bleached about 75% of the
CA3-region and the maximal efficacy of the DDC is
between 15 min and 1 h 45 min. After this delay, the ef-
fect of DDC gradually decreases with time.
Therefore, during the behavioural experiments, we
will keep a delay of 15 min between the moment of in-
jection and the beginning of the behavioural test.
Moreover, the mean duration of the learning procedure
in the spatial open-field and in the object recognition task
never exceeds 50 min, so we are sure to have a maximal
effect of the DDC during behavioural tests.
 G. Stupien et al. / Neurobiology of Learning and Memory 80 (2003) 32–41
Fig. 2. Relationship between index measure (relative difference in
staining intensity between injected and non-injected hippocampal re-
gion) and survival time following intrahippocampal injection of DDC
(0.25 ll; 200 mM). Each delay represents the median of five mice.
3. Experiment 2: Spatial open-field
3.1. Materials and methods
3.1.1. Surgery
Mice were anaesthetised with chloral hydrate
(400 mg/kg) and placed in a stereotaxic apparatus (Da-
vid Kopf Instruments) with mouse adapter and lateral
bars. The head skin was cut longitudinally and bilateral
guide cannulae (0.56 mm in diameter) were fixed on the
calvarium with dental acrylic. The following coordinates
with lambda and bregma in the same horizontal plane
were used: posterior to bregma, 1.6 mm; lateral to
midline, 2 mm; and 1.5 mm beneath the skull surface.
The subjects were then left in their home cage for a re-
covery period of 7–8 days.
3.1.2. Drugs
To block the hippocampal CA3-region, we used the
diethyldithiocarbamate and drug injected animals were
always compared to control mice injected with the same
volume of Ca-ethylenediamine tetraacetic acid (Ca-
EDTA). Ca-EDTA is also a chelating agent but cannot
penetrate plasma membranes and does not chelate the
heavy metals within axonal buttons (Danscher, Shipley,
& Andersen, 1975). Both drugs were administered as
aqueous solutions (200 mM) and injected in a volume of
0.25 ll/side. The injection time was 2 min 30 s for each
side and the needle was left in place for an additional
60 s to allow diffusion.
3.1.3. Reaction to novelty apparatus
The apparatus (Fig. 3) was a circular open-field,
60 cm in diameter with a 30-cm-high wall made of grey
plastic material, and a painted white floor divided into
sectors by black lines. The open-field was surrounded by
a visually uniform environment except for a conspicuous
striped pattern, 30 cm wide and 36 cm high (alternating
35
Fig. 3. Schematic representation of the apparatus and the object
configuration over successive sessions. a, the open-field is initially
empty (S1); in the three subsequent sessions (S2–S4), it is filled with
objects in a particular configuration (b). In S5, two objects are dis-
placed (spatial novelty) (c).
1.5 cm-wide vertical white and black bars) attached to
the wall of the field. A video camera above the field was
connected to a video-recorder and a monitor.
Five objects were simultaneously present in the open-
field: (A) a chromium-plated parallelepiped (7
4

4 cm) with holes irregularly distributed on the sides and
the top; (B) a plastic cone on a transparent cylinder base
(diameter of the section 8 cm; height 6 cm); (C) two grey
iron rectangles (10
3 cm) forming a 90° angle, inserted
on a rectangular plastic base (7
5 cm); (D) a black
Plexiglas cylinder (height: 10 cm; diameter: 5 cm); (E) a
black and grey spool (height: 9 cm; diameter of the top
5 cm) on a square base (6
6 cm).
3.1.4. Procedure
The training procedure was the same as that used in
previous experiment (Roullet et al., 2001; Sargolini
et al., 1999). Mice were tested during the first half of the
light period (between 9:00 a.m. and 2:00 p.m.). They
were individually submitted to four successive 6-min
sessions, separated by a 3-min delay, during which
subjects were returned to their home-cage. During ses-
sion 1 (S1) mice were placed in the empty open-field to
let than familiarise with the apparatus and to record the
36 G. Stupien et al. / Neurobiology of Learning and Memory 80 (2003) 32–41
baseline level of locomotor activity. During sessions 2–4
(S2–S4), the objects were placed as in Fig. 3. In session
5, the objects were displaced relatively to the original
arrangement. Object E replaced object A, which was
itself placed at the periphery of the apparatus, so that
the initial square arrangement was changed to a poly-
gon-shaped arrangement.
The injections were made by inserting an injection
needle (0.25 mm in diameter) into the guide cannula,
connected with a polyethylene tubing to a 1 ll Hamilton
syringe. Mice were remained in their cages during the
injection time.
For the learning procedure (Fig. 4), injections were
made 15 min before the beginning of the first session
(S1) and the delay between the last session of habitua-
tion (S4) and the spatial change (S5) was 3 min. For the
memory consolidation and recall procedures, mice were
submitted to the spatial change 24 h after the last session
of habituation. The injection was made immediately
after S4 for the memory consolidation procedure and
15 min before the spatial change for the recall procedure.
3.1.5. Data collection and statistics
Data collection was performed using video-record-
ings (for details see Save, Foreman, & Buhot, 1992). In
the first session, two behavioural parameters were re-
corded: leaning and locomotor activity, assessed by
counting the number of sectors crossed by each animal
while moving in the open-field. From sessions 2–5, ob-
ject exploration was evaluated by the time spent by the
animal in contact with an object. A contact was defined
as the subjectÕs snout actually touching an object.
For the acquisition experiment, all mice were ran-
domly assigned to one of two treatment groups. For the
memory consolidation and recall experiments, mice were
assigned to treatment groups according to their activity
and habituation performances during sessions 1–4, in an
attempt to avoid any pre-treatment differences between
Fig. 4. Experimental design using diethyldithiocarbamate (DDC) to
study the different phases of memory in non-associative spatial task.
groups. An ANOVA was used to verify the homogeneity
of performances in the two groups of mice before the
treatment.
In session 1 (without object), the effects of DDC in-
jection on locomotor and leaning were evaluated by an
independent t-test. Habituation to object exploration
during sessions 2–4 was performed by a factorial
analysis of variance, with session (three levels) as within-
subject factor, and treatment (two levels) as between-
subject factor.
In session 5, the spatial arrangement of the objects
was modified. In order to avoid an experimental bias
due to preference for one object, response to spatial
change was assessed by comparing the mean time in
contact with the objects belonging to each category
(displaced and non-displaced) in session 5 minus the
mean time spent in contact with the same object cate-
gory in session 4. A positive value indicates a renewal of
exploratory activity and a negative value an habituation
to this set of objects. A difference between exploration of
displaced and non-displaced object, indicates that mice
are able to compare the new arrangement of objects with
a stored representation of the previous one. This would
indicate memory of spatial relationships. For this com-
parison ANOVA analysis was performed with ‘‘object
category’’ (two levels) as within-subject factor, and
treatment (two levels) as between-subject factors.
In all experiments, statistical analysis did not reveal
any sex-related effect.
3.1.6. Histology
At the completion of the experiment, mice were sac-
rificed by an overdose of chloral hydrate. The brains
were removed and frozen at )20 °C. Cannula place-
ments were determined by examination of serial coronal
sections (40 lm) stained with thionine.
3.2. Results
Only animals showing a correct placement of cannula
were included in the statistical analysis. When DDC or
Ca-EDTA injections overflowed on the CA1 or CA4
regions, mice were removed from the analysis to be sure
to block only the CA3-part of the dorsal hippocampus
in this experiment.
3.2.1. Acquisition
Injection: 15 min before session 1; spatial change: 3 min
after session 4. During session 1, DDC did not induce
any change in sector crossing ½F ð1; 14Þ ¼ 1:26; p ¼ :231
nor in leaning ½F ð1; 14Þ ¼ 0:258; p ¼ :620 . The habit-
uation curves for the two groups are shown in Fig. 5A.
The ANOVA revealed no treatment effect ½F ð2; 28Þ ¼
:708; p ¼ :50 , no session effect ½F ð2; 28Þ ¼ :878; p ¼ :42
and no treatment
session interaction ½F ð1; 14Þ ¼ :05;
p ¼ :82 .
 G. Stupien et al. / Neurobiology of Learning and Memory 80 (2003) 32–41 37

Fig. 6. Memory consolidation: Reactivity to spatial change of mice

after immediate post-training focal administration of EDTA or DDC
at 0.25 ll/side into the hippocampal CA3-region. The histograms il-
lustrate the mean time (SEM) spent exploring the displaced objects
(DO) and non-displaced objects (NDO) in S5 minus the time spent
exploring the same class of objects in the last session of habituation
(S4).

and a significant interaction between the two factors

½F ð1; 14Þ ¼ 5:48; p ¼ :035 .

3.2.3. Recall
Fig. 5. (A) Objects exploration during the three habituation sessions.
The curves represent the mean time (SEM) spent exploring each
Injection: 15 min before session 5; spatial change: 24 h
object before the spatial change. (B) Acquisition: Reactivity to spatial after session 4. During the first session and during
change of mice after pretraining focal administration of EDTA or habituation sessions, ANOVA revealed no significant
DDC at 0.25 ll/side into the hippocampal CA3-region. The histograms pre-treatment differences between the two groups.
illustrate the mean time (SEM) spent exploring the displaced objects Fig. 7 shows the effects of EDTA and DDC injections
(DO) and non-displaced objects (NDO) in S5 minus the time spent
exploring the same class of objects in the last session of habituation
expressed as a difference between S5 and S4 for the two
(S4). object categories. Administration of DDC just before
session 5 seems not to affect detection of a spatial
change. The ANOVA revealed a main object effect
Fig. 5B shows the time spent exploring the displaced ½F ð1; 14Þ ¼ 23:37; p < :001 but no significant treatment
objects (DO) and the non-displaced objects (NDO) ex- effect ½F ð1; 14Þ ¼ 158; p ¼ :697 or interaction between
pressed as the difference between S5 and S4 in each the two factors ½F ð1; 14Þ ¼ 1:13; p ¼ :305 .
group. DDC injected mice re-explored the displaced
objects less than the control mice. The ANOVA revealed
a significant effect of object category ½F ð1; 14Þ ¼ 31:61;
p < :001 and a significant treatment
object interaction
½F ð1; 14Þ ¼ 4:79; p ¼ :046 but no significant treatment
effect ½F ð1; 14Þ ¼ :238; p ¼ :63 .

3.2.2. Memory consolidation

Injection: immediately after session 4; spatial change:
24 h after session 4. During the first session and during
habituation sessions, ANOVA revealed no significant
pre-treatment differences between the two groups.
In this experiment, when we injected DDC immedi-
ately after the habituation sessions, the reaction of mice
to the spatial change decreased in comparison to the
control group. The two-way ANOVA of the difference Fig. 7. Recall: Reactivity to spatial change of mice after focal ad-
ministration of EDTA or DDC at 0.25 ll/side into the hippocampal
between the time spent exploring the two categories of
CA3-region 24 h after S4 and just before S5. The histograms illustrate
objects in sessions 5 and 4 (Fig. 6) revealed no significant the mean time (SEM) spent exploring the displaced objects (DO) and
treatment effect ½F ð1; 14Þ ¼ :726; p ¼ :409 but a sig- non-displaced objects (NDO) in S5 minus the time spent exploring the
nificant main object effect ½F ð1; 14Þ ¼ 36:36; p < :001 same class of objects in the last session of habituation (S4).
38 G. Stupien et al. / Neurobiology of Learning and Memory 80 (2003) 32–41

4. Experiment 3: Object recognition 1 and 2, evaluated by the time spent by the animals in
contact with the different objects. A contact was defined
4.1. Materials and methods as the subjectÕs snout actually touching an object.
A habituation index was also calculated as the difference
4.1.1. Apparatus in time spent exploring the two objects during the ses-
Test objects were presented in a PVC square arena sion 1 minus the time spent exploring the object during
(50
50 cm), with walls 30 cm high painted grey and a session 2.
white floor divided by black lines into 16 identical sec- Statistical analyses were performed using an ANOVA
tors. The apparatus was placed in a separate room with a within–between design. For the mean exploration
containing no conspicuous features, illuminated by a time, the repeated factor was object (2 levels) and the
white light (60 W) and surmounted by a video camera between factor was treatment (2 levels). For the habit-
connected to a video recorder and a monitor. uation index, we used a one-factor ANOVA design
Two different objects, named A and B, made of glass (treatment: two levels).
or metal were employed in this task, and their weight
was such that they could not be displaced by animals: 4.2. Results
(A) a glass cylinder (height: 7 cm; diameter: 6 cm) in-
serted on a square plastic base (7
7 cm) and (B) a 4.2.1. Acquisition
metallic disc (dept: 4 cm; diameter: 7.5 cm) fixed on the Injection: 15 min before session 1; object change: 15 min
plastic base. after session 1. Fig. 8A illustrates the level of exploration
of the two objects during sessions 2 in the control and in
4.1.2. Procedure the DDC injected mice. In this experiment, during
The general procedure consisted of three different session 2, DDC injected and control mice explore more
phases: a familiarisation phase, a sample phase and a the new than the familiar object. During session 1,
choice phase. On the first day mice were individually ANOVA showed no significant object effect ½F ð1; 14Þ ¼
submitted to a single familiarisation session of 10 min, :301; p ¼ :592 , no treatment effect ½F ð1; 14Þ ¼ :001;
during which they were introduced in the empty arena,
to become familiar with the apparatus and to record the
baseline level of locomotor activity. On the second day
animals were submitted to a single 10 min session, dur-
ing which two identical objects (A1 and A2) were placed
in a symmetric position from the centre of the arena,
each 10 cm from the side wall. After a delay during
which mice returned to their home-cage, they were re-
introduced to the arena and exposed to two objects, a
familiar object (A3) and a new object (B), placed at the
same location of the sample stimuli. The familiar object
was a triplicate copy of the sample used in session 1, to
avoid olfactory trails. The role (sample or new object) as
well as the location of the two choice objects during
session 2 was counterbalanced between mice and across
sessions.
Two different procedures were used in the present
study: an acquisition procedure, in which a 15 min delay
was introduced between the two sessions, and a memory
consolidation procedure, in which the delay was
lengthened to 24 h.
For the acquisition procedure, the injections were
made 15 min before the beginning of the first session.
For the memory consolidation procedure, the injection
was made immediately after the first session and mice
were submitted to the probe trial 24 h later. Fig. 8. (A) Exploration of objects after pretraining focal administra-
tion of EDTA or DDC at 0.25 ll/side into the hippocampal CA3-re-
gion. (B) Exploration of objects after immediate post-training focal
4.1.3. Data collection and statistics
administration of EDTA or DDC at 0.25 ll/side into the hippocampal
Data collection was performed using video-record- CA3-region. The histograms illustrate the mean time (+SEM) spent
ings and the observer was always blind to treatment. exploring the different category of objects (familiar and new) during
The basic measure was object exploration during sessions session 2.
 G. Stupien et al. / Neurobiology of Learning and Memory 80 (2003) 32–41
p ¼ :993 and no interaction between the two factors
½F ð1; 14Þ ¼ 2:22; p ¼ :158 . During session 2, ANOVA
revealed an important object effect [F ð1; 14Þ ¼ 24:92;
p < :001 but no treatment effect ½F ð1; 14Þ ¼ 1:09;
p ¼ :314 and no treatment
object interaction
½F ð1; 14Þ ¼ :097; p ¼ :760 . For the habituation index,
there is also no treatment effect ½F ð1; 14Þ ¼ 1:911;
p ¼ :188 .
4.2.2. Memory consolidation
Injection: immediately after session 1; object change:
24 h after session 1. Fig. 8B shows the mean time of
exploration of the two objects during session 2 in the
two groups of mice. As in the acquisition experiment,
DDC injected mice seem always able to detect and to
react to the introduction of the new object after a delay
of 24 h. In session 1, ANOVA showed no significant
object effect ½F ð1; 13Þ ¼ 4:38; p ¼ :056 , no treatment
effect ½F ð1; 13Þ ¼ :722; p ¼ :411 and no interaction be-
tween the two factors ½F ð1; 13Þ ¼ :395; p ¼ 541 . Dur-
ing session 2, mice spent more time in contact with the
new object ½F ð1; 13Þ ¼ 16:14; p < :001 and DDC
treatment did not change the general exploration of the
two objects ½F ð1; 13Þ ¼ 1:15; p ¼ :30 and the detection
of the new one ½F ð1; 13Þ ¼ :29; p ¼ :59 . No treatment
effect was found on the habituation index
½F ð1; 13Þ ¼ 2:692; p ¼ :125 .
5. Discussion
The purpose of this study was to investigate the role
of hippocampal CA3-region in the different phases of
memorisation of two non-associative tasks. The spatial
open-field is designed to estimate the ability of mice to
encode spatial relationships among discrete stimuli. In
our study, control mice injected by EDTA, reacted
strongly to the spatial configuration change in the three
procedures used in the spatial open-field. Our results
therefore confirm that mice are able to detect a change
in the spatial configuration of objects just after or 24 h
after sessions of habituation and than to encode spatial
relationships among objects (Roullet & Lassalle, 1990;
Roullet et al., 2001; Sargolini et al., 1999). In the object
recognition task, control mice investigated more the
novel than the familiar complex object just after the
session of habituation but also 24 h after in the memory
consolidation experiment. In this task, EDTA injected
mice discriminate the two objects and are able to iden-
tify the familiar object exactly like rats (Ennaceur &
Delacour, 1988; Ennaceur, Neave, & Aggleton, 1996).
We therefore conclude that these two tasks with the used
procedures, seem to be appropriate to study the different
phases of spatial or non-spatial memory in mice.
In the first experiment, we have shown that a DDC
injection into hippocampal CA3-region produces a
39
maximal bleaching of the neo-Timm coloration between
15 min and 1 h 45 min. This duration of DDC action in
mice is in accordance with literature data on rats (Holm
et al., 1991; Lees et al., 1998). Since our different
behavioural procedures last about 45 min, we are sure
that mice are always on the DDC effect during learning
sessions. In addition, with this short duration of action
of the DDC, we have not acted on the total duration of
the memory consolidation but only on the first phase of
the memory consolidation (approximately during 1 h
30 min).
The first aim of our experiments was to determinate if
hippocampal CA3-region is specifically involved in
spatial learning. Blocking this area produced an im-
pairment of the ability of mice to react to the spatial
change but did not produce an effect on the object rec-
ognition task during the different phases of learning.
The finding that the hippocampus is not involved in
recognition memory is consistent with previous studies
using fornix lesion (Bussey, Duck, Muir, & Aggleton,
2000; Ennaceur et al., 1996, 1997). It is important to
note that CA3-region blockage produces the same
spatial deficit as when blocking completely the dorsal
hippocampus. As already seen in the introduction,
dorsal hippocampus is involved specifically in spatial
learning in the radial maze (Jarrard, 1983; Olton et al.,
1978), in the Morris water maze (McDonald & White,
1994; Morris et al., 1982) and also in the spatial open-
field task. In this last task, a bilateral lesion of the
anterior part of the hippocampus (Save et al., 1992) or a
fimbria–fornix lesion (Sargolini et al., 1999) impairs the
detection or the reaction to the spatial change without
impairing the reaction to the non-spatial change when
we introduce a new object in the open-field. An intact
neural pathway from the entorhinal cortex through the
hippocampus to the subcortical structures innervated by
the fimbria–fornix is necessary for the performance in a
spatial task (Jarrard, 1978; OÕKeefe, Nadel, Keightley,
& Kill, 1975; Olton et al., 1978). Therefore, reversible
inactivation of the CA3-region in our experiment or
destruction of the CA3 pyramidal cells (Handelmann &
Olton, 1981) certainly interrupts this pathway, by re-
moving one target of information from the entorhinal
cortex and induces a deficit in spatial learning.
The second aim of this study was to investigate the
role of the hippocampal CA3-region in the different
phases of spatial memory formation. Our results show
that the hippocampal CA3-region is involved in the
acquisition but not in the recall of spatial information as
seen previously in the Morris water maze using mossy
fibres inactivation by DDC (Lassalle et al., 2000) or
using kainic acid lesions of CA3 pyramid cells of the
anterior hippocampal part (Handelmann & Olton,
1981). The main result of this study is that the hippo-
campal CA3-region is also involved in the first spatial
information memory consolidation process. In fact, with
40 G. Stupien et al. / Neurobiology of Learning and Memory 80 (2003) 32–41
our massed procedure, we are sure to work only on the
first information memory consolidation and not on the
reconsolidation after reactivation of the memory trace
(Przybyslawski et al., 1999; Nader et al., 2000; Sara,
2000). Two possibilities exist to explain the impairment
of memory consolidation immediately after a DDC in-
jection. First, this treatment could have provoked a
blockage of the information pathway from entorhinal
cortex to fimbria–fornix. But this blockage happens af-
ter the end of acquisition, there is therefore necessarily a
flow of information through the CA3-region during the
consolidation. Second, one part of the spatial informa-
tion treatment might be executed by this region and had
been interrupted by the DDC injection. This second
hypothesis is reinforced by other experiments on the
differential activation of the immediate early gene, the c-
fos, during memory consolidation. For example, after a
standard eight-arm radial maze learning, CA3-region (as
other hippocampal subfields, CA1, dentate gyrus, and
subiculum) showed an increase in c-fos activation 90 min
after the end of learning sessions (Vann, Brown, Erich-
sen, & Aggleton, 2000). In another task, the operant
task, Bertaina-Anglade, Tramu, and Destrade (2000)
have shown that this c-fos activation depends on the
animalÕs learning level. Indeed, after the first learning
session, a c-fos activation is observed specifically in the
CA3-region and this activation appears after each
learning session. On the other hand, the CA1-region is
only activated during the second learning session and
the dentate gyrus from the second to the last learning
session. In these experiments, all c-fos activations are
found again one hour after the last learning session and
not 2 or 3 h after. These results show first, that there is a
neural activation, measured by fos-staining, in CA3-re-
gion during the memory consolidation phase. Second,
this activation of c-fos is important from the first
training session (that is during the first memory con-
solidation) and this point is in accordance with the re-
sults of our study.
In conclusion, the present results, obtained using a
massed procedure, show that the hippocampal CA3-re-
gion is involved in acquisition but also in memory
consolidation in a spatial non-associative task. As a next
step, it seems important to investigate whether this re-
gion is involved in the memory consolidation of any
kind of spatial learning and not only in a non-associa-
tive spatial task.
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