Edited by: Benno Roozendaal, Radboud University Nijmegen Medical Centre, Netherlands
Reviewed by: Antonio Armario, Universitat Autònoma de Barcelona, Spain; Walter Adriani, Istituto Superiore di Sanità, Italy
*Correspondence: Anne Nosjean, Centre de Neuroscience Paris Sud, Université Paris Sud 11 and Centre National de la Recherche Scientifique UMR 8195, Bâtiment 446, 15 Bd Clémenceau 91405 Orsay, France e-mail:
This article was submitted to the journal Frontiers in Behavioral Neuroscience.
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Adult C57BL/6J mice are known to exhibit high level of social flexibility while mice lacking the β2 subunit of nicotinic receptors (β2−/− mice) present social rigidity. We asked ourselves what would be the consequences of a restraint acute stress (45 min) on social interactions in adult mice of both genotypes, hence the contribution of neuronal nicotinic receptors in this process. We therefore dissected social interaction complexity of stressed and not stressed dyads of mice in a social interaction task. We also measured plasma corticosterone levels in our experimental conditions. We showed that a single stress exposure occurring in adulthood reduced and disorganized social interaction complexity in both C57BL/6J and β2−/− mice. These stress-induced maladaptive social interactions involved alteration of distinct social categories and strategies in both genotypes, suggesting a dissociable impact of stress depending on the functioning of the cholinergic nicotinic system. In both genotypes, social behaviors under stress were coupled to aggressive reactions with no plasma corticosterone changes. Thus, aggressiveness appeared a general response independent of nicotinic function. We demonstrate here that a single stress exposure occurring in adulthood is sufficient to impoverish social interactions: stress impaired social flexibility in C57BL/6J mice whereas it reinforced β2−/− mice behavioral rigidity.
Social interactions involve highly integrative and adapted behaviors to make coherent decisions in specific environmental contexts. Social interactions are altered in numerous psychiatric pathologies, such as anxiety (Lukkes et al.,
The prevalence of stress-induced pathologies has increased tremendously in industrialized countries, to the extent that they have become a major global health problem. An individual's ability to prevent, correct, or escape stressful situations to reach a state of personal well-being reflects the overall health of society. Numerous studies have addressed the short- and long-term effects of chronic stress on brain physiology and plasticity, on cognitive function in adults, and on cognitive function during childhood and adolescence (Veenema,
Mice that lacked the β2 subunit of nicotinic receptors (β2−/− mice) did not exhibit cognitive defects (Zoli et al.,
Social behaviors are complex behaviors that require sequential choices to achieve an action and integrate both emotional and motivational processes (Adolphs,
In a recent work, we validated a software highlighting social behaviors (MiceProfiler, de Chaumont et al.,
Male C57BL/6J mice and β2−/− knockout mice purchased from Charles Rivers Laboratories (L'Arbresle Cedex, France) were used in the present study. β2−/− mice were generated from a 129/Sv Embryonic Stem cell line as previously described (Picciotto et al.,
All experimental procedures were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC), EU Directive 2010/63/EU, Decree N 2013-118 of February 1st, 2013, and the French National Committee (87/848). Experiments were conducted in order to reduce the number of mice used and their level of discomfort. C57BL/6J and β2−/− mice, 10–11 weeks old, were group-housed (four mice/cage; size of the cages L: 26.5 cm, l: 16.5 cm, H: 13.5 cm) at their arrival to acclimatize to the animal facilities (food and water
The social interaction task was performed in a transparent Plexiglass box (L: 50 cm × l: 25 cm × H: 31 cm) located in an experimental room with light level set at 100–110 Lux by indirect white bulbs. For each experiment, the floor of the cage was covered with clean sawdust. The box was placed under a camera connected to a computer located outside of the experimental room.
Social behaviors were studied in dyads of male mice composed by an Isolated Host mouse (IH) and a Social Visitor mouse (SV) brought together for the first time. IH mice were individually housed 4 weeks before the social interaction task, while SV mice remained group-housed. IH mice (either C57BL/6J or β2−/− mice) were submitted or not to an acute stress. SV mice were always not stressed C57BL/6J male mice. The choice of 4 weeks social isolation was made after pilot experiments. All behavioral experiments were performed from 9.00 a.m. to 2.00 p.m. (i.e., in the lit phase of the dark/light cycle). Each isolated animal randomly assigned to the stressed groups was placed for 45 min in a Falcon® tube. After 5 min in its home cage, IH mouse was placed in the experimental box for 30 min exploration. Then, a SV mouse was gently introduced in the box, in the corner opposite to the IH mouse. The delay from the beginning of the stress to the end of the social task (i.e., 84 min) was compatible with expression of the c-fos protein in the PFC (Weinberg et al.,
Social interactions between SV and IH mice were videotaped and analyzed using Mice Profiler software (de Chaumont et al.,
Chronograms and density graphs were built to illustrate the evolution of the quantitative data in the course of mice dyad interactions. They show behavioral variation of a specific event within an animal and between animals over time. As such, they allow to perceive the stability or instability of given behaviors. For each mouse or each dyad, chronograms depict the frame-by-frame occurrence and duration of a particular event, and thus provide an immediate visualization of its frequency. Density graphs (built from chronograms) represent the temporal evolution of the density of a given event, i.e., the sum of the time in which an event is happening vs. the total time of the bin (grouped here in bins of 30 s). As such, they represent the same information as chronograms but averaged for all mice of a specific group. We also built transitional graphs to illustrate the probability of incidence of one event or posture on the next or the previous one in a sequence.
Finally, we scored dominance and aggressiveness by off line manual analysis. Dominance index was defined as the number of “paw control” (i.e., the number of times IH mouse placed its forepaw on the head or on the back of the SV mouse). Aggressiveness was indexed by the cumulated number of tail rattling and attacks over the 4 min of the experiment. Latencies to first attack and first tail rattling were also scored.
Biochemical measures of plasma corticosterone were carried out in a pseudo random order by an experimenter blind to mice genotype and stress conditions. Experiments were performed in the same time scale than behavioral ones (10 a.m.–1.30 p.m., i.e., in the lit phase of the dark/light cycle), animals being treated similarly than for behavioral experiments (see Supplementary Figure
Normal distribution (assessed by Shapiro-Wilkonson test) and equality of variance (evaluated by equal variance test) were first tested using Sigmaplot 12.0. When both were statistically significant, main factor effects and interactions were tested with Two-Way ANOVAs followed by Fisher LSD Method tests when appropriate. When normality and/or equal variance were not statistically significant, Kruskal-Wallis One-Way ANOVA then Mann-Whitney
For similar number, β2−/− mice spent significantly more time in close contact a1 with their partner than C57BL/6J mice [genotype effect:
No statistical changes in number and duration for oral-oral contacts (a2 event) were detected (
In regards IH oral-genital contacts with SV mouse (a3 event), β2−/− mice made significantly more such contacts than C57BL/6J mice [
SV mouse made more oral-genital contacts a4 with β2−/− mice than with C57BL/6J mice (
Concerning side-by-side contacts, a5 event increased in number (
Overall, events that were increased in β2−/− mice as compared to C57BL/6J were reduced in β2−/− animals after stress and reached C57BL/6J values.
All contact events occurred during the course of the task for all groups of mice (Supplementary Figure
Thus, most contact subtypes were more numerous and lasted longer in β2−/− mice than in C57BL/6J mice. Overall, exposure to restraint stress did not alter contact behaviors of C57BL/6J mice but drastically reduced them in β2−/− mice. They acquired a C57BL/6J social like behavior despite different evolution with time.
The event b1 (IH mice behind SV mice) occurred similarly in C57BL/6J and β2−/− mice [no genotype effect:
The events b2 (SV mice behind IH mice) occurred more frequently in β2−/− mice as compared to C57BL/6J mice [genotype effect:
In all groups, b1 event was similarly stable all along the experiment. In contrast, b2 event gradually increased over time in both C57BL/6J and β2−/− mice and remained steadily low in stressed C57BL/6J mice. This suggested that C57BL/6J and β2−/− mice, but not stressed C57BL/6J mice, became used to have their partner behind them.
Thus, all groups of IH mice similarly and continuously preferred to be behind SV mice overtime. Acute stress reduced the total time that mice spent in both positions only in C57BL/6J mice and seemed to prevent stressed C57BL/6J mice to accustom to have their partner behind them.
To target specific dynamic behaviors, we dissociated those initiated by SV mice (Table
No statistical changes were detected for number of c2 event (IH mouse approached by SV mouse). SV mice approached more quickly a β2−/− than a C57BL/6J mouse (
SV approaches terminated by a social contact (c3 event) were significantly less numerous (
Finally, as regards c4 event, the only statistical change concerned its number that was significantly increased after stress in β2−/− mice compared to not stressed β2−/− mice (
As a matter of fact, SV actions that were significantly reduced in presence of a β2−/− mice compared to C57BL/6J mice (c3, c4), increased after stress to reach C57BL/6J mice values.
Approaches of SV mice (c2, Supplementary Figure
Here, we showed that SV approaches, shorter in presence of a β2−/− mouse, were restored after stress. This revealed that the social approaches of SV mice were influenced by the β2−/− genotype and their emotional status.
Concerning the second order events (d4, d5), no genotype effect [
Finally, the third order events d6 was only affected in C57BL/6J mice after stress. In these mice, stress produced a decreased of d6 number (
For all groups, d2 events (Supplementary Figure
The only difference between C57BL/6J and β2−/− mice concerned the first order events (approaches and follow behaviors) that were more numerous and lasted longer in β2−/− animals. In C57BL/6J mice, acute stress led to an increase in first order events and to a decrease in second and third ones indicating a reduction in the complexity of behavioral sequences in this strain. In β2−/− mice, stress did not alter the second and third order events but reduced and therefore normalized first order events such as approaches and escapes (Table
The number of stop events of SV mice (e1) was unaffected by the genotype [
For similar duration, no genotype effect was detected in the number of IH stop events (e2) between C57BL/6J and β2−/− mice [
Event e1 increased in the first minute of the task in not stressed mice and in the last minute in stressed ones. In contrast, e2 event drastically decreased after the first minute of the task to remain steadily low in all groups except in C57BL/6J mice. This indicates that stop behaviors of the host mice were important feature of an organized and flexible social repertoire in C57BL/6J mice and that they were compromised in animals exhibiting flexibility defects, whether β2−/− mice or stressed animals.
Thus, C57BL/6J mice spent more time at rest than β2−/− mice. Acute stress reduced this time in C57BL/6J mice but had no effect in β2−/− animals. Also, SV mice spent less time at rest in presence of stressed C57BL/6J mice but not when facing a β2−/− mice (stressed or not). Thus, SV mice behavior was only influenced by C57BL/6J behavior.
The number of paw control, on one hand, and of tail rattling and attacks, on the other hand, were manually quantified to respectively evaluate the dominance and the aggressiveness of IH mice during social interaction. SV mice never showed tail rattling and never attacked IH mice.
Paw control | 5.45 ± 0.98 | 18.75 ± 3.60 |
15.56 ± 2.84 |
19.29 ± 4.09 |
Tail rattling | 0.00 | 6.06 ± 3.05 |
0.00 | 12.21 ± 6.00 |
Attacks | 0.00 | 3.94 ± 1.99 |
0.00 | 9.07 ± 4.35 |
IH mice | 240.00 ± 0.00 | 149.00 ± 29.72 |
240.00 ± 0.00 | 119.57 ± 46.45 |
IH mice | 240.00 ± 0.00 | 169.63 ± 22.14 |
240.00 ± 0.00 | 132.71 ± 41.80 |
The number of paw control was significantly higher in β2−/− mice than in C57BL/6J mice (
Thus, restraint stress increased dominance only in C57BL/6J mice, and induced aggressiveness in both mouse genotypes. Aggressiveness was not due to previous mice isolation since it did not exist in not stressed mice. As such, it was imputable to stress and was independent of nicotinic system.
Statistical correlations we performed to identify putative relationships between the different events of the mice social repertoire within each group of mice. Supplementary Figure
With C57BL/6J mice, SV mice actions did not participate in the generation of contact or complex events (
Thus, we evidenced drastic differences in the arrangement of social events between C57BL/6J and β2−/− mice. Acute stress deconstructed the links existing between the different events of a given behavioral class and between the various classes of the social repertoire in both genotypes. This occurred even if there is no quantitative difference in the social repertoire and in the emergence of aggressiveness in stressed C57BL/6J and stressed β2−/− mice (Table
Transition graphs were built to investigate how the various events fit together during the social interaction task. They represent the probability of a given event to be preceded or followed by one or several events and provide complementary information from quantitative data. Indeed, d3 event, poorly represented in number or time in all groups (Figure
In all groups, stops of the SV mouse (e1, black arrows) triggered all main contacts, postures, and dynamic events of the dyads. It suggested that stops of the SV mouse triggered fixed behaviors whatever the IH mouse it faced. This occurs even if SV mice spent less time at rest in presence of a stressed than a not stressed C57BL/6J mouse (Figure
SV mice were always C57BL/6J group-housed not stressed mice. Therefore, independently of the genotype or the emotional status of the social partner, common transitions between key behavioral events were centered on stops of the SV mice. Moreover, host mice from the different groups, which were always previously isolated and habituated to the environment, shared two key behavioral events: stops and follow behaviors.
In addition to common transitions described above, the different groups of mice share or not some transitions (see below).
C57BL/6J stop events (e2) triggered close contact (a1), relative position (b2), and follows (d3) events. In contrast, stop events of β2−/− mice (e2), which lasted significantly less than that of C57BL/6J mice (Figure
Thus, stop events played a crucial role in the initiation of numerous different behaviors in C57BL/6J mice. In contrast, follow behaviors appeared to play a fulcrum role in β2−/− animals.
In C57BL/6J mice, long sequences existed, while acute stress induced shorter ones. In not stressed mice, stop events (e2) triggered several sequences (blue arrows) while in stressed mice, e2 event which lasted significantly less than in C57BL/6J mice (Figure
Interestingly, some strings only observed after stress in C57BL/6J animals existed in β2−/− mice. Also, d3 event in stressed C57BL/6J mice appeared crucial to initiate behavioral sequences as seen for β2−/− mice. Thus, these results complemented our quantitative data showing some similarities of social behavior between stressed C57BL/6J and not stressed β2−/− mice.
Overall, acute stress shortened behavioral sequences in C57BL/6J mice and dramatically triggered aggressive behaviors (Table
Some transitions observed in stressed and not stressed β2−/− mice existed in stressed C57BL/6J mice (Figure
Overall, acute stress in β2−/− mice induced behavioral transitions common to the C57BL6 strain or specific to all stressed animals. These data and quantitative results (see Table
Plasma corticosterone levels were measured as described in Supplementary Figure
C57BL/6J | 1.21 ± 0.48 | 12.73 ± 0.98 |
|
C57BL/6J Stress | 13.42 ± 2.42 |
11.85 ± 1.79 |
|
β2−/− | 1.11 ± 0.79 | 10.23 ± 1.72 |
|
β2−/− Stress | 20.10 ± 2.75 |
13.55 ± 1.66 |
The present study was performed to determine the immediate impact of acute restraint stress on social interactions in adult male mice and to investigate the contribution of neuronal nicotinic receptors (nAChRs) in these effects. C57BL/6J mice were used, because they were known to exhibit high sociability (Sankoorikal et al.,
We found that, in all 4 types of dyads, SV stops always occurred at the starting point of contact events, postures, or dynamic event, regardless of the emotional status or genotype of its partner. In addition, for all dyads, IH stops and follow events were key points in the social repertoire.
We first confirmed here previous data as β2−/− mice made longer contacts and more numerous follows, spent less time at rest and were more dominant (Granon et al.,
We revealed a crucial importance of dominance behaviors: in C57BL/6J mice dominance was associated with a large panel of social contact but this was not the case in β2−/− mice, for which dominance seemed an isolated compound (Figure
In summary, the present study showed that each mouse genotype exhibited a specific complex social behavioral pattern and developed distinct social strategies. It highlighted major behavioral difference between C57BL/6J and β2−/− mice, hence the important role of the cholinergic nicotinic system in the social strategy used by animals to organize their behavior. The differences between C57BL/6J and β2−/− mice in this task may rely on a different trade-off between exploitation and exploration: C57BL/6J mice would favor exploration of various options, including social contact when confronted to a novel environment, and β2−/− mice would favor exploitation of the social reinforcement. Favoring exploitation over exploration has been shown to be accompanied by rigid behaviors (Cohen et al.,
Although SV mice were not subjected to stress procedure, it indirectly altered their behavior: it shorted their stops and their tolerance when facing a stressed C57BL/6J mouse, indicating that SV was influenced by the emotional status of its partner. This can be due to the increase of follows, dominance and aggressiveness exhibited by stressed mice. This adaptation to their social partner, in turn, deeply modified their entire social repertoire as seen in transition graphs. In another social paradigm (3 chambers), Yang et al. (
Stress had three main consequences in IH C57BL/6J mice: it impoverished the social repertoire, increased dominance, and fostered aggressive behaviors. The social impoverishment was illustrated by the reduction of complex sequences with concomitant increase in single events such as approaches and follows, the shortening of some contact events and of mutual tolerance (Table
We reported that single restraint stress increased dominant behaviors by more than three-fold in C57BL/6J mice, and dramatically altered their consequences. Social dominance is a normal behavior (Blanchard et al.,
We showed that aggressiveness and dominance were dissociated social features that were never correlated, as previously reported (Coura et al.,
Social experiments reported by others were mostly performed during the light phase of the cycle, although some experiments were conducted during the dark phase. However, it has been reviewed recently that sociability in rodents can be efficiently evaluated in the light phase (Yang et al.,
In conclusion, stressed C57BL/6J mice managed social contact with a novel social partner by reorganizing their behavior; moreover, they reduced or eliminated complex sequential behaviors. Interestingly, the dominance level in stressed C57BL/6J mice increased to the levels observed in β2−/− mice. This finding suggested that stress may alleviate behavioral inhibition mediated by the cholinergic system (Picciotto et al.,
Stressed β2−/− mice acquired C57BL/6J-like social behavior. That is, in β2−/− mice, stress drastically reduced social contacts to the levels observed in C57BL/6J mice, and it normalized approaches, escapes and the number of stops. However, stress had no effect on tolerance, follows, or complex behaviors. After stress, follows were totally disconnected from all behavioral events (Figure
In conclusion, stress in β2−/− mice produced a dramatic reduction in the social repertoire, a clustering of behavioral sequences, emergence of aggressiveness, and apparent social normalization.
C57BL/6J and β2−/− mice exhibited similarly low baseline levels of corticosterone. Also, their levels of plasma corticosterone were comparable after immobilization stress and after stress followed by novelty exploration. This reinforced the idea that the behavioral differences reported here between stressed and not stressed C57BL/6J and β2−/− mice could not be accounted for by plasma corticosterone absolute levels. However, variation in these levels differed in both genotypes: it remained stable whether animals explored novelty or were only stressed in C57BL/6J mice, whereas it significantly dropped after novelty exploration despite previous stress in β2−/− mice. β2−/− mice seemed to be more reactive to restraint stress than C57BL/6J mice. As mentioned above, this provided evidence that β2−/− mice were more sensitive to their internal status than to external cues, leading, in consequences, to a larger variation in their corticosterone levels in between different behavioral conditions. The comparison between stressed C57BL/6J and stressed β2−/− mice showed that acute stress tended to stereotype social actions of both mouse genotypes, and thus, it made both of them less flexible. This finding goes along with recent data obtained in humans, which showed that cold stress imposed before an instrumental task promoted habits (Schwabe and Wolf,
The present exhaustive behavioral analysis revealed that acute stress in adulthood was sufficient to trigger a marked increase in aggressiveness in both C57BL/6J and β2−/− mouse genotypes. Because β2−/− mice lacked nicotinic receptors, these results showed that aggressive bursts were not linked to nAChR function. We further showed that they were not linked to plasma corticosterone levels. In contrast, stress dramatically increased dominance only in C57BL/6J mice; this finding supports the notion that dominance and aggressiveness are unrelated processes, with only dominance depending on functional nAChRs. Acute stress impoverished mouse social interactions by altering the relationship between behavioral classes, and the classes altered depended on whether the mice exhibited a flexible or pathological social pattern. Thus, stress induced behavioral rigidity in “socially competent” animals, and it worsened rigid behavior in pathological models.
Taken together, these data showed that a unique experimental framework could tease apart behaviors in mice that represent components of the vicious stress-aggression cycle proposed in humans (Craig,
Substantial contributions to the conception of the work: Sylvie Granon, Jean-Christophe Olivo-Marin, to the design of the work: Anne Nosjean, Sylvie Granon, to the acquisition analysis: Anne Nosjean, Arnaud Cressant, Fabrice de Chaumont, Frédéric Chauveau, to the interpretation of data: Anne Nosjean, Sylvie Granon, Arnaud Cressant, Fabrice de Chaumont, Frédéric Chauveau. Drafting the work: Anne Nosjean, Sylvie Granon; critical revision of the work: Anne Nosjean, Sylvie Granon, Arnaud Cressant, Fabrice de Chaumont, Frédéric Chauveau, Jean-Christophe Olivo-Marin. Final approval of the version to be published and agreement for all aspects of the work: Anne Nosjean, Sylvie Granon, Arnaud Cressant, Fabrice de Chaumont, Frédéric Chauveau, Jean-Christophe Olivo-Marin.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This work was supported by the Centre National de la Recherche Scientifique (CNRS, UMR 8195), by the Université Paris Sud 11 (Chaire d'Excellence to Sylvie Granon). It was also supported in part by a grant from the Agence Nationale de la Recherche.
The Supplementary Material for this article can be found online at: