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). After surgery and before testing, each animal was rst handled
(10 min) daily for three consecutive days. Then it was deprived of water 48h before
the beginning of training. During training of the following days, the rats were given
water ad libitumfor 30min per day at 6:30 pm. European guidelines on procedures
for animal experimentation were followed, and all efforts were made to minimize
the animals suffering and to reduce the number of animals used while complying
with statistical constraints.
2.2. Lesion surgeries
All rats were anaesthetized with an intraperitoneal (ip) administration of pen-
tobarbital (60mg/kg, i.p.). Once anaesthetized, they were given an intramuscular
injection of an antibiotic (Extencilline; 0.3mL/kg) and were subsequently placed
on a stereotaxic apparatus. Their scalp was opened longitudinally with a scalpel
and little holes were drilled in the skull at locations where a needle had to be
introduced into the brain to perform the ibotenate infusions. The ibotenate solu-
tion (5g/L) was infused at the coordinates indicated in Table 1 at a speed of
0.25L/min. After each infusion, the needle was left in place for 4min before being
slowly retracted. In the sham-operated rats, the needle was descended into the
brain at each of the coordinates used for the lesion, but no infusion was performed.
After surgery, the scalp was sutured and all rats were placed in a heated cage until
complete recovery from anesthesia. Behavioral training was started 4 weeks after
surgery.
2.3. Apparatus and training procedure
The olfactory training apparatus was a rectangular box made of wire mesh
(30cm30cm50cm). A conical odor port (1.5cm in diameter, 0.5cm above the
oor) was drilled horizontally through a triangular wedge of Plexiglas, mounted
in one corner of the cage. A circular (1cm diameter) water port in the shape of a
well was placed directly above the odor port. Responses to the odor presentation
Fig. 1. Representative photographs illustrating lesions following NeuN staining in the groups of SHAM(A, C, E) and lesioned (B, D, F) rats. B and D showtypical examples of
lesions in the dorsolateral striatumand in the dorsal hippocampus, respectively. Dashed lines indicate the limits of the damaged area. E and F indicate that rostral regions of
the hippocampus were not damaged after lesions of the dorsal hippocampus (F) as compared to a sham-operated rat (E). Scale bars at the bottomright of photos B, D and F
correspond to 1M.
96 M. Jacquet et al. / Behavioural Brain Research 239 (2013) 94103
Fig. 2. Schematic representation of the smallest (light gray) and largest (dark gray) extent of dorsal hippocampal (left) and dorsolateral striatal (right) lesions in the rats kept
for statistical analyses. Numbers above the drawings indicate the distance (in mm) of each section fromBregma (according to Paxinos and Watson, 1998).
were monitored by a photoelectric circuit. Two ashlight bulbs which could be
turned on and off, as the testing conditions required, were placed outside the
cage, one on each side of the odor and water ports, 10cm above the oor. Indi-
vidual odors were delivered by forcing clean air (0.7 bars) through one of two
1 L Erlenmeyer asks that contained 500mL of water mixed with 0.2% of chemi-
cal odorants (jasmine vs. strawberry) from Sigma Inc. Non-odorized air, between
each odor presentation, was delivered by sending air through a ask that contained
only water. Odorized and clean air streams were passed individually through tubes
that were put through the back of the sound-attenuating chamber and attached to
the odor port. Water was delivered using a gravity-fed system, and passed through
a valve which, when opened, allowed 0.1mL of water to be released into the water
port.
All experiments were conducted simultaneously in four olfactory cages to
ensure that, in each experiment, representatives from each group were trained at
the same time, and thus under identical conditions [10,24]. Animals were trained
to make two odor-reward associations. Each odor had to be associated with a spe-
cic reward, one arbitrarily designated as positive and the other as negative. One
session was made of 60 trials using a successive Go or No-Go paradigm. Individual
trials (S
+
or S
) was 10s.
Correct responses, therefore, corresponded to Go before the 10s presentation for
one odor had elapsed (S
+
) and No-Go to the other (S
C) over
5 min. The brains were extracted, postxed for 2h in the same xative (4
C), and
transferred into a 0.1M phosphate-buffered 20% sucrose solution for about 48h
(4
laten-
cies (Fig. 3C) showed a dramatic impairment in the DL STRIATUM
group. Indeed, converselytotheir SHAMcounterparts, lesionedrats
were unable to make correct associations on S
+
and S
stimuli.
In the SHAM group, correct associations were observed from the
fourth session onwards [F(1,18) 15.39; p<0.001]. The DL STRIA-
TUMgroup also showed a slight gradual decrease in the time taken
to respond to both stimuli across the ve sessions. SHAM rats
showed a continuous decrease in latencies of response to S
+
stimuli
throughout the training session while for S
the latencies for the negative odor. Only the SHAM group started to make
signicant associations fromsession 3 onwards.
disrupted the establishment of stimulus-response associations
[13,27] or altered the habit-based memory system [28]. How-
ever, in agreement with these authors, dorsal striatum lesions
marginally impair the initial learning of odor discrimination. Fur-
thermore, before a decit became apparent, the presentation of
three successive discrimination problems was required. Comp-
ton [29] suggested that the dorsal striatum is necessary for the
mediation of stimulus-response learning while the hippocampus is
necessary to mediate expression of place learning (in their experi-
ments); these processes can occur simultaneously and in parallel.
In our current experiment, dorsolateral striatumlesioned rats were
both impaired in cumulative time and in odor-reward associa-
tions, whereas hippocampal lesioned rats were able to withhold
a response during the ITI but showed unable to make correct
stimulus-response associations. To master the timing in this task
a stimulus-elicited response is not required and the dorsolateral
striatum rats were impaired by responding randomly during the
ITI. So, in addition to stimulus-response impairments, it seems that
striatumlesioned rats did not gure out howto use the task to get
only the positive reward and to get it rapidly. This decit might be
interpreted as consequent to an impairment of suppressing auto-
matic responding.
Inrodents, lesions altering the medial part of the dorsal striatum
usually impair goal-directed learning. Damage to the dorsolateral
striatum is described as impacting habit learning (for a review,
see [30]). Research on skill learning and cognitive control is often
guidedbyamultiplememorysystems framework. This framework
usually contrasts declarative memory, which provides exible and
explicite access to semantic and episodic content and requires con-
scious awareness, with memory for howto implement procedures
(i.e., how to perform a sequence of actions). These procedures
can become automatic and subconscious [31]. Based on the recent
reviewby Liljeholmand ODoherty [30], the impairments reported
herein after lesions of the dorsolateral striatumcould be the result
of impaired procedure learning and consequently reect a proce-
dural memory dysfunction.
Althoughseveral studies showa dissociationbetweenthe forms
of memory related to the striatum (procedural) and those related
to the hippocampus (declarative), there is also evidence pointing
to the involvement of the caudate nucleus in declarative memory
[32,33]. For instance, Ben Yakov and Dudai [34] performed fMRI
experiments in humans in which they measured brain activity
time-locked to the offset of short narrative audiovisual episodes.
They found that a set of brain regions, most prominently the
hippocampus and the caudate nucleus, both bilaterally exhibited
memory-predictive activity that was time-locked to the stimu-
lus offset. The hippocampus and caudate nucleus are involved in
registeringintegratedepisodes tomemoryas cohesiveinternal rep-
resentations. It cannot be excluded that in our rats the lesions of
the dorsolateral striatum disabled a similar process and thus pre-
vented learning by affecting also the declarative-like component of
the task.
We would nevertheless privilege an alternative explanation.
The fact that procedural memory decits prevented the acquisi-
tion of the association, and thus a declarative-like item, could be
questioned from a view considering encoding in the SPI system
proposed by Tulving in humans [35]. This systemplaces procedural
memory at the lowest hierarchical level, semantic memory above,
the highest level being that of episodic memory. From this func-
tional hierarchy of encoding processes, it can be derived that, in
tasks requiring a serial engagement of procedural and declarative-
like memory processes, as seems to be the case for the task used
in the current study, a decit affecting the lowest system should
have consequences on the efciency of the higher one. This is one
possible interpretation of our observations in rats with lesions of
the dorsolateral striatum. In our study both the hippocampus and
striatumwere involved in learning and memorizing the meaning of
each olfactory cue. It is thus possible, given the type of task which
weused, that thedorsolateral striatumandthedorsal hippocampus
had to contribute to task acquisition in a sequential order. First the
dorsolateral striatummight have been required to master the pro-
cedural component of the task, mainly during the two rst sessions.
Then, thehippocampus might havetakenover theacquisitionof the
odor-reward associations, and thus the declarative-like items. In
other words, before being able to make correct associations engag-
ing the hippocampus, rats had to learn the timing sequence of
the task, for which the engagement of the dorsolateral striatum
might be crucial. Indeed, learning to respond only when an olfac-
tory cue is presented and not to respond during the inter-trial delay
(when only a neutral air is on) required a tuned control of the loco-
motor neural system to engage into going or refrain from going
(engage into No-Go behavior). Then, in a second phase, a choice
has to be made in order to respond only to the positively-rewarded
M. Jacquet et al. / Behavioural Brain Research 239 (2013) 94103 101
S1
0 1 2 3 4 5 6
0
5
10
15
20
C
U
M
U
L
A
T
I
V
E
T
I
M
E
(
s
e
c
.
)
S2
0 1 2 3 4 5 6
0
2
4
6
8
10
S3
0 1 2 3 4 5 6
0
2
4
6
8
10
S4
0 1 2 3 4 5 6
0
2
4
6
8
10
S5
0 1 2 3 4 5 6
0
2
4
6
BLOCK OF TRIALS
D HIPP
SHAM
S=SESSION
Fig. 7. Mean cumulative time in blocks of 10 trials (SE) recorded during sessions S1 to S5. Both groups showed a time decreasing over sessions. No signicant difference
was observed between SHAMand D HIPP groups. See also legend to Fig. 6.
cue. Whenthe striatum-dependent learningis preventedbystriatal
lesions, the acquisition of the hippocampus-dependent selection
of the correct odor to engage in an appropriate response might
become impossible. Previous results using this olfactory associa-
tive task showed that 5-HT4 receptors are substantially involved
in the modulation of learning and memory processes [24]. Prior to
the rst training session, the injection of a selective 5-HT4 receptor
antagonist (RS 67532) was followed by an increase in cumulative
time, demonstrating that treated rats were more active at gur-
ing out how to get the reward and delayed the upcoming trial
by responding during the ITIs on the rst session. This behavior
allowed themto learn the protocol timing faster and consequently,
once the procedural aspect of the task was acquired, treated rats
startedto make consistent cuerewardassociations during the sec-
ond session while control rats did not start until the third one. The
injections of the partial agonist (RS 67333) prior to the rst session
had no signicant effect, whether on the rst or subsequent ses-
sions, and only a learning decit was observed. One interesting
point, in this study, was that injections administered prior to the
rst or thirdtrainingsessiongaverisetooppositeeffects. Afacilitat-
ing effect on learning and memory was found on session 3 when RS
67333hadbeeninjectedprior to this session, andanopposite effect
when the injection occurred prior to the rst session. The same
procedure with antagonist RS 67532 injections was followed by a
learning and memory performance decit on session 4 when the
injection occurred before the third session, and a facilitatory effect
on session 2 when it occurred before the rst session. The opposite
effects obtained in these experiments with the partial agonist and
antagonist canbe relatedto the sub-categories of memory involved
inthis task. Decreasing the activationof the hippocampal systemby
the use of a 5-HT4 receptor antagonist at the onset of training facili-
tates procedural memory, andthe inverse seems true for the partial
agonist. By contrast, once the timing component of the task (proce-
dural memory) has been learned (after the rst two sessions), the
injection of the partial agonist facilitates hippocampal processing
andconsequentlyimproves memory for the associations (reference
memory, related to declarative memory in humans); the opposite
reasoning applies to the antagonist. As a whole, fromthese exper-
iments, it seems that procedural learning comes rst in this task
and is crucial for acquiring subsequent declarative-like learning.
When declarative-like and procedural memory processes are
investigated in laboratory rodents, they are often considered as
operating in dual and sometimes even competing memory sys-
tems. In rats trained for response learning in the cross-maze task,
Packard and McGaugh [36] showed that the initial approach of the
task engaged a hippocampus-dependent declarative-like memory
systemacquiring knowledge about place. Only over task repetition
was there a gradual shift towards a striatum-dependent response
memory system. Approaches and tasks such as the ones used by
Packard and McGaugh posit the declarative-like memory as a pre-
requisite for response memory to be established over behavior
repeats (see also [37,38]). In the test situation used in our current
experiments, the cognitive operations required for task acquisi-
tion seem to occur according to an inverse schedule: only when
102 M. Jacquet et al. / Behavioural Brain Research 239 (2013) 94103
S1
0 1 2 3 4 5 6
0
1
2
3
4
5
6
7
8
9
10
L
A
T
E
N
C
Y
(
s
e
c
.
)
S2
0 1 2 3 4 5 6
0
1
2
3
4
5
6
7
8
9
10
S3
0 1 2 3 4 5 6
0
1
2
3
4
5
6
7
8
9
10
S4
0 1 2 3 4 5 6
0
1
2
3
4
5
6
7
8
9
10
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
S5
0 1 2 3 4 5 6
0
1
2
3
4
5
6
7
8
9
10 ***
BLOCK OF TRIALS
D HIPP S +
D HIPP S -
SHAM S +
SHAM S-
S=SESSION
Fig. 8. Mean latencies in block of 10 trials (SE) recorded during sessions S1 to S5. S
+
are the latencies for the positive odor and S