Modulation of the Mouse Prefrontal Cortex Activation by Neuronal Nicotinic Receptors during Novelty Exploration but not by Exploration of a Familiar Environment
Cerebral Cortex May
Modulation of the Mouse Prefrontal Cortex Activation by Neuronal Nicotinic Receptors during Novelty Exploration but not by Exploration of a Familiar Environment
Jean-Pierre Bourgeois 2 3
Vannary Meas-Yeadid 1 2
Anne-Marie Lesourd 2 3
Philippe Faure 0 2
Ste´ phanie Pons 0 2
Uwe Maskos 0 2
Jean-Pierre Changeux 0 2
Jean-Christophe Olivo-Marin 0 2
Sylvie Granon 0 2 4
0 Unite ́ de Neurobiologie Inte ́ grative des Syste` mes Cholinergiques, Unite de Recherche Associee Centre National de la Recherche Scientifique 2182, De ́ partement des Neurosciences, Institut Pasteur , 75015 Paris , France
1 Unite ́ d'Analyse d'Images Quantitative, Unite de Recherche Associee Centre National de la Recherche Scientifique 2582, Institut Pasteur , 75015 Paris , France
2 The Author 2011. Published by Oxford University Press. All rights reserved. For permissions , please
3 Unite ́ de Ge ́ ne ́ tique Humaine et Fonctions Cognitives, Unite de Recherche Associee Centre National de la Recherche Scientifique 2182, De ́ partement des Neurosciences, Institut Pasteur , 75015 Paris , France
4 Current address: unite ́ mixte de recherche Centre National de la Recherche Scientifique 2195, Universite ́ Paris Sud XI, Centre de Neuroscience Paris Sud , 91405 Orsay , France
Organization of locomotor behavior is altered in mice knockout for the b2 subunit of the nicotinic receptor-b22/2 mice-during novelty exploration. We investigated the neuronal basis of this alteration by measuring activation of the immediate early gene c-fos in the brains of wild-type (WT) and b22/2 mice after exploration of a novel or a familiar environment. Results show 1) no constitutive difference between WT and b22/2 mice in c-fos gene expression in any brain region, 2) novelty exploration triggered activation of the hippocampus and the reward circuit while exploration of a familiar environment produced increased activation in the amygdala, and 3) in b22/2 mice, exploration of novelty, but not familiarity, induced an increase in activation in the prelimbic prefrontal cortex (PFC) compared with WT mice. c-Fos immunoreactivity after different stages of learning in a maze increased similarly in the prelimbic area of both WT and b22/2 mice, while their performance differed. In WT mice, exploration of a novel environment triggered an increase in c-Fos expression in the reward circuit and the hippocampus, while in b22/2 mice, the amygdala and the motor cortex were additionally activated. We also highlight the role of nicotinic receptors during activation of the PFC, specifically during free exploration of a novel environment.
c-Fos; nAChRs; novelty; prefrontal cortex; reward circuit
The b2 subunit--containing nicotinic cholinergic
receptors—b2*nicotinic cholinergic receptors (nAChR)—are
transmembranal allosteric proteins, widely expressed throughout
the mammalian brain (for review, Changeux and Edelstein
2005). Previous investigations have shown that mice bearing
the null mutation for the b2 subunit of the nicotinic
cholinergic receptor—b2–/– mice (Picciotto et al. 1995)—have
increased locomotor activity and decreased exploratory
behavior when confronted with a novel environment (Granon
et al. 2003; Granon and Changeux 2006; Besson et al. 2007).
Locomotor sequences were disorganized during novelty
exploration (Maubourguet et al. 2008), but not when mice
were habituated to an environment (Wiklund et al. 2008). The
implication of the ventral tegmental area—VTA—and the
specific contribution of the cholinergic system in these
behaviors have been demonstrated by rescue experiments
using lentiviruses (Maskos et al. 2005; Avale et al. 2008). Since
the potential contributions of other related brain regions remain
elusive, here we explored other neuronal correlates of these
behavioral deficits using 3 distinct exploratory behavioral
situations: 1) the first involved free exploration in a novel environment;
2) the second involved repeated exploration of the same
environment, thus providing a control for processes associated
with familiarity versus novelty; 3) the third was designed to
compare more constrained exploration with free exploration.
In previous experiments (Granon et al. 2003), b2–/– mice
showed better spatial learning than wild-type (WT) mice
during the early learning stages. After a few days of learning,
however, performance of both strains were similar. We
assessed the neuronal bases of the behavioral performance in
WT and b2–/– mice using quantitative expression of the
immediate early gene c-fos, which reflects enhanced neuronal
physiological activity (Bisler et al. 2002; Staiger et al. 2002;
Majdan and Shatz 2006; Lim et al. 2009). The quantification of
c-Fos immunoreactive nuclei provides evidence of the regional
patterns of brain areas that are simultaneously activated in
specific cognitive/behavioral tasks.
Our results support the view that b2*nAChRs modulate the
prefrontal cortex (PFC) activity, specifically in flexible choice
Materials and Methods
Thirty-eight b2–/– mice and 38 C57Bl/6J—WT—mice were used in
these experiments. They were 4 months old males arriving from the
rearing facilities (Charles Rivers Laboratory, France) 2--3 weeks before
the experiment. Extensive genomic analysis has been carried out to
determine the percentage of C57BL6/J genetic background in the b2–/–
line that originally derives from a 129/Sv embryonic state cell line. Since
then, the line has been backcrossed more than 20 generations with the
WT C57BL6/J line, which is above the 10 backcrosses recommended by
the Banbury conference (Banbury Conference on Genetic Background
in Mice 1997). Using more than 400 genomic markers, the b2–/– line
was confirmed to be at more than 99.99% C57BL/6J.
The animals were treated according to the ethical standards defined
by the Institut Pasteur and Centre National de la Recherche Scientifique
for animal health and care in strict compliance with the EEC
Two weeks before being tested in any behavioral test, mice were
placed in individual cages in a ventilated and temperature- and
humiditycontrolled room with a 12/12 light:dark cycle (light at 8:00 AM).
For the open field experiment (open field novelty exploration WT n = 10,
b2–/– n = 10; familiar open field exploration WT n = 10, b2–/– n = 9; and no
open field WT n = 10, b2–/– n = 10), they received food pellets and water ad
libitum. For the maze learning experiment (WT n = 8, b2–/– n = 9), they
were food deprived for 10 days before the beginning of any learning
procedure. Once the weight of the mice reached 85% of their free feeding
weight, habituation of the learning procedure started.
Open Field Exploratory Behavior
As described previously (Granon et al. 2003; Maskos et al. 2005; Besson
et al. 2007), the open field consists of an opaque white plastic circular
tank 1 m in diameter and 40 cm in height, located in an isolated room
with large and distinct distal cues permanently fixed to the walls. The
light was set at 100 Lux at the center of the open field. Each mouse was
transferred from its individual cage in a transport box, taken to the
experimental room, and from there to the center of the open field for
a unique session of 30 min (open field novelty condition). For the
familiar open field exploration, mice were treated as for the novelty
condition twice a day for 3 consecutive days. For the control condition
(no open field), the animals were taken from their home cage, placed in
the same transport box, transported to the experimental room, and
then taken back immediately to their home cage.
At the end of each session, the mouse was returned to its own cage
and perfused 90 min later. The open field was cleaned between each
The apparatus has been described in detail elsewhere (Granon et al.
2003). Briefly, it consists of 4 arms forming a cross. One arm contained
a food cup containing sucrose pellets at the end. The opposite arm had
a similar food cup filled identically but it was covered with a grid,
making the food unavailable. Two longer arms were used as starting
points. The goal of the task was to reach the food cup where the food is
available from 1 of the 2 pseudorandomly starting arms. The animals
were placed in the maze for 3 trials per day, with an intertrial interval of
2 min during which time the animals were returned to their home cage.
Behavioral Measures and Conditions
Open Field Exploratory Behavior
During the open field sessions, a video camera connected to a computer
equipped with a video tracking system (View-point, Lyon, France),
located above the open field, automatically recorded the locomotor
activity (total distance in centimeters) of each animal in absence of any
After stabilization of their weight to 85% of their free feeding weight,
mice were familiarized to the maze for 2 days for 15 min with food
scattered in every arm—maze visit condition—before the learning
protocol started. At that stage, all animals ate in the maze during the 2
first min. Learning consisted of 3 trials per day where animals were
gently placed in 1 of the 2 start arms. Performance was measured by
latency to reach the food (a maximum of 2 min was allowed). The
animals from each genotype were randomly and blindly assigned to the
5 learning phases: no exploration (animals were food deprived and did
not visit the maze), maze visit day 2 (animals visited and ate in the maze
but did not learn any specific location as food was scattered all over the
maze), learning day 5 (animals learn the food cup location for 5
consecutive days), learning day 10 (animals learn the food cup location
for 10 consecutive days), and learning day 15 (animals learn the food
cup location for 15 consecutive days).
At the end of each experimental condition, the animals were returned
to their home cage for 90 min to allow for the synthesis and transport
of c-Fos proteins to the nuclei of activated neurons. This interval (90
min) was selected as it is within the period of peak production
(between 90 and 120 min) for c-Fos protein after a specific, initiating
event (Bisler et al. 2002). The animals were then sacrificed with
pentobarbital and perfused transcardiaally with 150 mL of
phosphatebuffered saline (PBS), followed by 50 mL 4% paraformaldehyde
depolymerized in saline phosphate buffer. The brains were extracted
and postfixed overnight in the cold room. Sections (70 lm) were cut
throughout the entire brain using a vibratome (Leica). Residual free
aldehydes in the tissue were neutralized in PBS containing 50 mM
NH4Cl, 1 mM lysine, and 1 mM glycine. Endogenous peroxidases were
neutralized in PBS containing 3% H2O2 for 20 min. Brain sections were
treated for 30 min in a solution of PBS containing 0.1% Triton to
permeabilize the brain cells and 1% bovine serum albumin (BSA) and
5% normal goat serum (NGS) to saturate nonspecific binding sites. The
c-Fos proteins were immunolabeled in rotating vials at 4 C for 38 h. We
used a 1:8000 dilution of the purified polyclonal rabbit immunoglobulin
G (IgG) anti-human c-Fos (AB-5) human (Calbiochem #PC38) in PBS
with 1% BSA, 1% NGS, 0.05% NaN3 (sodium azide, Sigma S-2002), and
0.1% Triton. After 3 rinses (10 min each) in PBS, the first antibody was
tagged successively with purified and biotinylated goat anti-rabbit IgGs
(Vector BA-1000) diluted 1:600 in PBS containing 1% BSA and 1% NGS
for 2 h and then with an avidin--peroxidase complex (Vectastain E´ lite
PK 6100) for 30 min at room temperature. After several rinses in
Trisbuffered saline, peroxidase was revealed using H2O2 (33%) and
diaminobenzidine (Sigma D-5905) as the chromogen. The peroxidase
reaction was stopped after 3 min. Sections were mounted on
SuperFrost glass slides dehydrated in ethanol and coverslipped with
Regions of Interest
We quantified c-fos expression in 6 distinct brain regions: prelimbic
cortex (Prl), primary motor cortex (M1), cornu ammonis field 1 of the
hippocampus (CA1), basolateral nucleus of the amygdala (BLA),
nucleus accumbens (NuAcc), and the VTA. These regions are known
to be necessary for either spatial cognition, such as the hippocampus
(Poucet and Save 2005; Renaudineau et al. 2009) and the Prl (de Saint
Blanquat et al. 2010), or are part of a network which showed a different
role in b2–/– and WT mice (Maskos et al. 2005; Ballesteros-Ya´ n˜ez et al.
2010). We systematically sampled the median section of the whole
rostrocaudal extent of each of these brain structures (from bregma:
Prl +1.98 mm, M1 +0.62 mm, CA1 –3.28 mm, BLA –0.94 mm, NuAcc.
+1.34 mm, VTA –3.40 mm; Paxinos and Franklin 2004).
Image Acquisition and Quantification of c-Fos--Positive Nuclei
Sections were viewed under a Nikon Eclipse1000 light microscope, and
images were acquired using a digital camera (Nikon DXM 1200). A 310
Plan Apo objective gave both good resolution of c-Fos immunoreactive
(c-Fos+) nuclei and identification of the cytoarchitectonic fields. The
focus was set on the upper face of each section before digitization. The
final magnification, calibrated with a micrometer object slide (Zeiss),
scaled 100 lm in the tissue to 80 pixels in the images. Digitized images
were then quantified on a Sun station using an in-house developed
multiresolution algorithm (Olivo-Marin 2002). The contour of each
region of interest (ROI) was drawn on the monitor screen for each
digital image. The software automatically calculated the surface of the
ROI, converted the number of c-Fos--positive nuclei into gray spots, and
computed their density per square micrometer. The
immunocytochemical background was eliminated from the quantification process
by a denoising algorithm using a threshold value that is image and level
dependent and can be computed automatically from the data
(OlivoMarin 2002). This program makes it possible to select and count cells
automatically without experimenter bias (counts were conducted
without knowledge of the group assignments). In order to derive
accurate, absolute cell counts, it would be necessary to use
stereological methods (Coggeshall and Lekan 1996), but the goal of
the present study, as it is the case in other work (e.g., Albasser et al.
2010), was to compare relative numbers of activated cells between
groups. In each coronal section, we sampled both ipsi- and contralateral
regions separately and then averaged the density of the spots for each
structure per animal. The results were expressed as dSPOTS per square
millimeter of cerebral tissue.
For open field experiments, the behavioral and c-Fos quantification data
were analyzed with analysis of variance (StatView) with genotype and
conditions (when applicable) as between-subject main factors or with
Statistical correlations (Z score correlation, StatView) were performed
concerning the number of c-Fos+ neurons between the different brain
areas for each experimental condition of the open field experiment and
between the number of c-Fos+ neurons and the distance covered in the
open field to control for a putative locomotor effect.
For the maze learning experiment, due to a limited number of animals
in each learning stage (n = 3 in each group per stage), we conducted
nonparametric Mann--Whitney test to assess the genotype effect.
Open Field Novelty Exploratory Behavior
For the open field exploration experiment, statistical analysis
(t-test) conducted on distance covered during novelty
exploration showed a significant genotype effect (tdf = 18 = 4.01,
P = 0.0008). These results, in agreement with previous studies
(Granon et al. 2003; Besson et al. 2007; Avale et al. 2008;
Maubourguet et al. 2008), validate the use of these mice for the
present study. The results showed that b2–/– mice have an
hyperactive phenotype, covering more distance (Fig. 1A)
compared with WT mice when exposed to a novel environment.
b2–/– and WT Comparison in Basal Condition—No
In baseline condition (no exploration), regional c-fos
expression was similar in b2–/– and WT mice in all ROIs (Fig. 1B,C).
Statistical analysis showed no difference between the 2
genotypes for Prl (F < 1, not significant [NS]), NuAcc (F < 1,
NS), VTA (F1,18 = 2.4, P = 0.1, NS), CA1 hippocampus (F < 1, NS),
basolateral amygdala (F1,18 = 1.5, P = 0.24, NS), and motor
cortex M1 (F1,18 = 1.2, P = 0.3, NS).
Novelty Exploration in WT Mice
When WT mice were exposed to a novel open field for 30 min,
they showed a significant increase in density of c-Fos+ nuclei
compared with basal levels (no exploration groups, Fig. 1B) in
the PFC (F1,18 = 5.8, P = 0.03), the VTA (F1,18 = 7.1, P = 0.02), the
NuAcc (F1,18 = 21.2, P = 0.0002), and the hippocampus (F1,18 =
10.5, P = 0.005). No increase in c-fos levels was observed in the
amygdala (F1,18 = 1.07, P = 0.31) and the motor cortex (F1,18 = 2.3,
P = 0.15).
Novelty Exploration in b2–/– Mice
In b2–/– mice, as in WTs, there is a significant increase in c-fos
expression triggered by novelty exploration in the PFC
(F1,18 = 9.8, P = 0.006), the VTA (F1,18 = 6.6, P = 0.02), the
NuAcc (F1,18 = 19.9, P = 0.0003), and the hippocampus (F1,18 =
40.2, P < 0.0001) (Fig. 1C).
In addition to what is observed in WTs, we also found
an increase in c-fos expression in the amygdala (F1,18 = 7.3, P =
0.01) and the motor cortex (F1,18 = 13.9, P = 0.002) in b2–/–
mice following exploration of the novel environment.
These results suggest that exploration of a novel
environment is sufficient to induce a significantly greater activity than
the baseline condition in the amygdala and the motor cortex in
b2–/– mice but not in WTs.
We performed correlation analyses between the number of
c-Fos+ neurons in different brain areas. In WT mice, there were
no correlation between the number of c-Fos+ neurons in any
brain region or between the number of c-Fos+ neurons and the
distance covered during exploration of the novel open field
(data not shown). By contrast, in b2–/– mice, the number of
c-Fos+ neurons in the prelimbic area of the PFC was correlated
with that of the NuAcc, CA1, amygdala, and motor cortex M1
(Table 1). The distance covered by the b2–/– mice was
correlated with the number of c-Fos+ neurons only in the
VTA (Table 1), suggesting that the higher locomotor level of
these mice is unlikely to account for increased cFos expression
in all brain regions. No significant correlation was observed
between locomotor activity and the number of c-Fos+ neurons
in WT mice for any brain region.
The same significant correlations were found between the
Prl and the amygdala and CA1 in animals not exposed to novelty
exploration (Table 2), suggesting that novelty exploration,
specifically in b2–/– mice, triggers correlated activation between
the Prl and the NuAcc and the motor cortex.
b2–/– and WT Mice Comparison during Exploration of
a Novel Environment
The only region in which expression of c-fos was significantly
greater in b2–/– mice as compared with WT was the prelimbic
area of the PFC (F1,18 = 6.6, P = 0.02) (Figs 1D and 2). WT and
b2–/– mice showed similar density of c-Fos+ nuclei in the VTA
Note: Plcx, prelimbic area of the prefrontal cortex.
***P \ 0.0001, **P \ 0.01, *P \ 0.05.
(F < 1, NS), the NuAcc (F1,18 = 2.3, P = 0.1, NS), the
hippocampus (F1,18 = 1.3, P = 0.3, NS), the amygdala (F < 1,
NS), and the motor cortex (F1,18 = 2.2, P = 0.2, NS).
Exploration of a Familiar Environment in WT and b2–/–
We measured c-fos expression after six 30-min sessions of
exploration of the same environment in order to compare brain
activations associated with novelty exploration and that
associated with familiarity to the environment. Behavioral data,
illustrated in Figure 3A, showed that both WT and b2–/– mice
habituated with repeated exposure (repetition effect: F5,85 =
30.4, P < 0.001 and interaction genotype 3 repetition: F5,85 =
2.47, P = 0.034). We found a significant genotype effect only
for the first (t = 2.7, degrees of freedom [df] = 17, P = 0.016),
second (t = 2.2, df = 17, P = 0.044), and sixth (t = 2.72, df = 17,
P = 0.014) exploration sessions.
c-fos expression data, illustrated in Figure 3B,C, showed
a similar increased activation in WT and b2–/– mice that was
specific to the amygdala after exploration of a familiar
environment when compared with exploration of a novel
environment (P < 0.0001). The level of c-fos expression did not
significantly differ in any other brain area after exploration of
a familiar environment compared with the novel environment
in both WT and b2–/– mice (all other P values > 0.05).
c-fos expression was similar in WT and b2–/– mice in all brain
areas after exploration of a familiar environment (all P values >
0.14), except the NuAcc where it is significantly increased in
b2–/– mice (t = 2.65, df = 17, P = 0.017).
b2–/– and WT Mice Comparison during the Different
Phases of Spatial Learning
We checked whether c-fos expression in the prelimbic area of
the PFC would be altered by a novel maze learning procedure.
The learning curve (Fig. 4A) showed a difference between b2–/
– and WT, as we previously observed (Granon et al. 2003), with
b2–/– mice learning faster than WT mice at day 5 (Z = 2.6, P =
0.009) and day 10 (Z = 2.0, P = 0.05) of learning, but at day 15 (Z
= 1.3, P = 0.18). We measured c-Fos+ nuclei in the prelimbic
area of WT and b2–/– mice (3 per behavioral stage and
genotype) after different stages of learning (Fig. 4B). We found
no difference between WT and b2–/– mice that were food
deprived but not tested in maze exploration (Z = 1.5, P = 0.13,
NS) or those given 2 days of exploration of the novel maze (Z =
0.2, P = 0.8). Neither was there a genotype effect at different
stages of spatial learning: day 5 (Z = 0.6, P = 0.5, NS), day 10 (Z =
0.2, P = 0.8), and day 15 (Z = 0.2, P = 0.8).
Due to the lack of difference between WT and b2–/– mice,
we pooled the 2 groups in order to check whether the c-fos
expression in the prelimbic area was altered by spatial
learning. We compared c-fos expression in the prelimbic
area after the different learning stages using Fisher tests (Fig.
4B). Statistical analyses show that c-Fos expression is
significantly greater in the prelimbic area of mice exploring
the maze as compared with mice only subjected to food
deprivation (P < 0.0001) and in mice in the first stage of
learning (day 5) compared with mice in the second stage of
learning (day 10, P = 0.03), indicating that exploration of the
novel maze is associated with an increased activation of the
Note: Plcx, prelimbic area of the prefrontal cortex.
***P \ 0.0001, **P \ 0.01, *P \ 0.05.
Our present results show that in WT mice, exploration of
a novel environment triggers an increase in c-fos expression in
the VTA, the NuAcc and the prelimbic area of the PFC, and the
hippocampus. In b2–/– mice, the amygdala and the motor
cortex were additionally activated.
Cerebral Activation during Exploration of a Novel
The implication of the hippocampus in spatial exploration and
learning has been recognized in rodents for decades (review in
Poucet and Save 2005), with place cell firing in area CA1 of the
hippocampus encoding spatial features needed for the memory
of a particular location (e.g., Cressant et al. 2002) and early
gene expression imaging its activation in different phases of
spatial memory (e.g., Jones et al. 2001; Armin et al. 2006;
Renaudineau et al. 2009). A similar form of activation has also
been shown in humans (Wittman et al. 2007). Field potential
and functional magnetic resonance imaging (fMRI) studies in
human and nonhuman primates reported hippocampal
activation triggered by the presentation of novel items compared
with familiar items (review in Ranganath and Rainer 2003).
Our results showed similar activation of the hippocampus
after open field exploration in WT and b2–/– mice, in
agreement with the fact that b2–/– mice show normal
habituation in spatial exploration (Granon et al. 2003; Wiklund
et al. 2008) but do not exhibit any spatial learning deficit or
hippocampal-like dysfunction until they age (Zoli et al. 1999).
The involvement of the different regions of the reward
circuit is more puzzling. Some previous works have suggested
that novelty would be rewarding and would constitute the basis
for novelty seeking, with novelty acting as an exploration bonus
(Krebs et al. 2009), possibly as novelty has a high biological
valence (Mesulam 1998), and is a natural potent feature of
external stimuli (Tulving et al. 1996). Recent fMRI data in the
human brain have implicated the substantia nigra/VTA system
(Wittman et al. 2007; Krebs et al. 2009), the NuAcc (Krebs et al.
2009), and the PFC (Daffner et al. 2000; Ranganath and Rainer
2003) in novelty exploration. However, there is little evidence
in rodent models that suggests specific brain regions
underlying the association between novelty exploration and
reward (Bevins and Bardo 1999; Dulawa et al. 1999). Here,
we provide new data giving evidence that exploration of
a novel environment is sufficient to activate major components
of the reward circuit (the VTA, the NuAcc, and the prelimbic
area of the PFC). In addition, we recently showed, in a different
behavioral context, that exploration of a novel object also
activates the same brain areas (Avale et al. 2011). Altogether,
the reward circuit seems to be particularly sensitive to novelty.
It is noteworthy that b2–/– mice show similar activation of
brain regions to WT mice with 2 notable exceptions: 1) in b2–/–
mice there is significant activation in the amygdala and motor
cortex following bouts of exploration compared with those not
exposed to the open field and 2) activation of the prelimbic
region is significantly increased in b2–/– mice compared with
WT mice. The elevated c-fos expression observed here in the
prelimbic cortical field of the WT mice might be tentatively
related to the significantly higher density of spines, bearing
mostly excitatory synapses, recently observed in this same field
(Ballesteros-Ya´ n˜ez et al. 2010). In the b2–/– mice, this density of
spines is significantly reduced (Ballesteros-Ya´ n˜ez et al. 2010),
while the expression of c-fos is significantly increased, in the
same field, as observed in the present study. We hypothesize
that this cognitive field would have a higher physiological
activity, as a result of the more active inputs from motor cortex
and amygdala as observed here, and/or a reduced inhibition.
The absence of the b2*nAChRs would specifically challenge the
functions of the associational cortical fields in novel situations.
Similar kind of compensatory mechanisms have been observed
in the human PFC. Indeed, effortful processing has been
associated with stronger activation of the cingulate cortex and
dorsolateral PFC in subjects for whom a cognitive task requires
higher attentional control (Cazalis et al. 2003).
Another interesting piece of data are that the activation of
the prelimbic region is positively correlated with that of the
NuAcc, M1, the hippocampus, and the amygdala only in b2–/–
mice after novelty exploration (see Table 1). Such pattern was
not found in WT mice for any behavioral conditions (data not
shown). In b2–/– mice, these correlations were not found after
exploration of a familiar environment (data not shown). It is
noticeable, however, that the correlations between the
prelimbic area and the hippocampus and the amygdala were
already significant in the baseline condition (Table 2).
There are established functional relationships between these
brain areas for the integration of motivational, memory, and
emotional information (O’Donnell and Grace 1995; Laroche
et al. 2000; Sesack et al. 2003). Specifically, data showing c-fos
activation indicate functional interactions between the PFC and
the NuAcc that would change as a function of the motivational
state of the animals (Moscarello et al. 2007). It is interesting
that these brain areas were activated in a correlational manner
in b2–/– mice exposed to novelty, as these mice have been
shown to exhibit altered motivational states (Picciotto et al.
1998; Maskos et al. 2005; Besson et al. 2007; Avale et al. 2011)
and increased response to novelty compared with WT mice
(Granon et al. 2003).
The absence of b2*nAChRs, which behaviorally triggers
increased locomotor response to novelty, may thus alter
homeostatic regulation within the emotional and motivational
circuits, suggesting that these receptors are important in
returning activity in these circuits to a steady state.
Cerebral Activation during Exploration of a Familiar
In contrast to familiar stimuli or context, novelty engages
a high level of attentional processing. These attentional
processes also rely on prefrontal activation, shown in both
humans and rodents (review in Kehagia et al. 2010). Activity in
cholinergic and noradrenergic neurons that project to the PFC
is sensitive to novelty, indicating that these neuromodulatory
systems are crucial for orienting attention to and enhancing
memory for novel stimuli (Sara et al. 1995; Yu and Dayan 2005).
Studies in human and nonhuman primates have shown that
familiarity results in a reduction in the population-level activity
(Ranganath and Rainer 2003); in our experiment, however,
activation triggered by exploration of a novel context did not
decrease when the context became familiar. Regarding the role
of attentional mechanisms in novelty processing as well as in
contextual memory, activation of the PFC in our experiments
may reflect attentional processing. Finally, it is likely that mice
maintain a high level of attention during each exploratory
session, independent of its level of familiarity, which may not
be the case in human subjects during laboratory experiments.
Alternatively, it could also reflect the triggering of memory
processes by activation of the same brain circuits as the ones
involved during the first exposure to the same environment.
b2–/– Mice Showed Significant Activation in the Amygdala
and the Motor Cortex as Compared with the Control
It is noticeable that in baseline conditions—no open field or no
maze exploration—b2–/– mice showed no difference to WT
mice for c-fos expression in any measured brain region.
However, lacking b2*nAChRs would provoke the recruitment
of the reward mesocorticolimbic circuit and additional brain
regions such as the amygdala and motor cortex. The
mechanisms of the crucial involvement of the nicotinic
receptors in the amygdala--prefrontal circuit, in relation to
the reward circuit, should be further investigated. It may help
understanding of the neural and molecular bases of the
relationship between novelty seeking and reward on one hand
and of novelty seeking and emotion on the other. Such
relationship may be crucial for understanding dysfunction that
gives rise to pathological states during which the processing of
emotional or motivational stimuli is altered, for example, in
depression, schizophrenia, posttraumatic stress disorder, or
addiction (Hains and Arnsten 2009; Koob 2009).
Cerebral Activation during Free Exploration versus
Constrained Learning: The Prelimbic Activation Is
Significantly Increased in b2–/– Mice as Compared with
There was no difference between WT and b2–/– mice in
expression of c-fos in the prelimbic region after spatial learning,
at any stage, although the performance differed. This suggests
that the role of nicotinic receptors in the PFC is specific to the
type of novelty processes engaged in open field free
exploration. It would be triggered by flexible behaviors
engaged by open choice, such as those available in an open
space with no explicit goal to be reached, as in the case of the
open field experiment. In a maze task driven by food
motivation, flexible behaviors are less required and exploration
can be viewed as being more constrained. Recent
electrophysiological recordings in the prelimbic PFC of rats showed
neuronal firing ‘‘before’’ choices were made, suggesting the
involvement of the prelimbic area of the PFC in prospective
behaviors (de Saint Blanquat et al. 2010). These results matched
our own showing the recruitment of the prelimbic area when
choices are open. Similarly, we show here the role of the
prelimbic area in the beginning of the maze learning task, but
this does not require b2*nAChRs. It is also a phase during
which attentional processes, known to be dependent on the
prefrontal/prelimbic activity (Granon et al. 1996, 2000), are
triggered. This dichotomy between the implication of
b2*nAChRs during the self-organization of behavior in open
field exploration versus their not being required during
exploration of a more constrained environment suggests
a specific role of the b2*nAChRs in flexible choices.
These results support the view that b2*nAChRs modulate
the dialogue between the PFC, which provides a cognitive
control of choices, and the reward, emotional and memory
systems, which provide cues for optimal motivational ranking.
Centre National de la Recherche Scientifique; Institut Pasteur;
ANR Neurobiologie and Psychiatrie 2005; Colle` ge de France;
Universite´ Paris Sud (chaire d’excellence to S.G.).
The authors thank Sabrina Davis for correction of English and Anne
Nosjean and Arnaud Cressant for helpful comments. Conflict of
Interest : None declared.
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