Anne Nosjean
email: anne.nosjean@universite-paris-saclay.fr

Sylvie Granon

Brain adaptation to acute stress : effect of time, social buffering and nicotinic cholinergic system

Keywords: Social Buffering, and Nicotinic Cholinergic System Acute stress, amygdala, c-fos protein, prefrontal cortex, time

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Social interactions involve high adaptive flexible behaviors that require the integration of internal and external cues to make coherent decisions in specific environmental contexts. Social interactions, altered in numerous psychiatric disorders, imply emotional and motivational processes and widely engage the prefrontal cortex (PFC) and the amygdala, in particular the basolateral nucleus of the amygdala (BLA) (see [START_REF] Showers | Social cognition: a look at motivated strategies[END_REF][START_REF] Adolphs | The neurobiology of social cognition[END_REF][START_REF] Adolphs | The social brain: Neural basis of social knowledge[END_REF][START_REF] Dalgleish | The emotional brain[END_REF][START_REF] Insel | How the brain processes social information: searching for the social brain[END_REF][START_REF] Bachevalier | The orbitofrontal-amygdala circuit and self-regulation of social-emotional behavior in autism[END_REF][START_REF] Lupien | Effects of stress throughout the lifespan on the brain, behaviour and cognition[END_REF][START_REF] Pessoa | Emergent processes in cognitive-emotional interactions[END_REF][START_REF] Stanley | Toward a Neural Basis for Social Behavior[END_REF][START_REF] Adolphs | Social and emotional neuroscience[END_REF][START_REF] Bickart | The amygdala as a hub in brain networks that support social life[END_REF][START_REF] Bicks | Prefrontal Cortex and Social Cognition in Mouse and Man[END_REF][START_REF] Janak | From circuits to behaviour in the amygdala[END_REF][START_REF] Demolliens | Social and asocial prefrontal cortex neurons: a new look at social facilitation and the social brain[END_REF]; for reviews). Besides the classical involvement of the autonomic nervous system, the hypothalamic-pituitary-adrenal axis and the hippocampus in stress responses, the PFC and the amygdala are also stress-related brain areas (Buijs et Van Eden 2000;[START_REF] Mcewen | Physiology and neurobiology of stress and adaptation: central role of the brain[END_REF][START_REF] Cerqueira | The stressed prefrontal cortex. Left? Right![END_REF][START_REF] Holmes | Stress-induced prefrontal reorganization and executive dysfunction in rodents[END_REF][START_REF] Lupien | Effects of stress throughout the lifespan on the brain, behaviour and cognition[END_REF][START_REF] Mora | Stress, neurotransmitters, corticosterone and body-brain integration[END_REF][START_REF] Mcewen | Stress effects on neuronal structure: hippocampus, amygdala, and prefrontal cortex[END_REF]Negrón-Oyarzo et al. 2016;[START_REF] Atrooz | Stress, psychiatric disorders, molecular targets, and more[END_REF]; for reviews). Among brain neuromodulators, acetylcholine plays a major role in social interactions. Brain cholinergic transmission mediated by nAChRs is also mainly involved in healthy and pathological cognition [START_REF] Graef | Cholinergic receptor subtypes and their role in cognition, emotion, and vigilance control: an overview of preclinical and clinical findings[END_REF][START_REF] Picciotto | Acetylcholine as a neuromodulator: cholinergic signaling shapes nervous system function and behavior[END_REF][START_REF] Hurst | Nicotinic acetylcholine receptors: From basic science to therapeutics[END_REF][START_REF] Levin | Complex relationships of nicotinic receptor actions and cognitive functions[END_REF][START_REF] Mineur | Multiple nicotinic acetylcholine receptor subtypes in the mouse amygdala regulate affective behaviors and response to social stress[END_REF][START_REF] Prado | Cholinergic circuits in cognitive flexibility[END_REF]Muramatsu et al. 2018;[START_REF] Záborszky | Specific basal forebrain-cortical cholinergic circuits coordinate cognitive operations[END_REF] and in stress regulation [START_REF] Mark | Inescapable stress enhances extracellular acetylcholine in the rat hippocampus and prefrontal cortex but not the nucleus accumbens or amygdala[END_REF][START_REF] Mora | Stress, neurotransmitters, corticosterone and body-brain integration[END_REF][START_REF] Paul | Interaction of basal forebrain cholinergic neurons with the glucocorticoid system in stress regulation and cognitive impairment[END_REF][START_REF] Picciotto | Mood and anxiety regulation by nicotinic acetylcholine receptors: A potential pathway to modulate aggression and related behavioral states[END_REF]. Notably, the β2, β4, and α7 subunits of the neuronal nicotinic cholinergic receptors (nAChRs) are broadly involved in social behavior [START_REF] Avale | Prefrontal nicotinic receptors control novel social interaction between mice[END_REF][START_REF] Salas | Abnormal social behavior in nicotinic acetylcholine receptor β4 subunit-null mice[END_REF][START_REF] Potasiewicz | 3-Furan-2-yl-N-p-tolylacrylamide, a positive allosteric modulator of the α7 nicotinic receptor, reverses schizophrenia-like cognitive and social deficits in rats[END_REF], Nosjean et al. 2018).

We previously performed exhaustive analyses of social behavior and brain activity in C57BL/6J mice (B6 mice) and in mice lacking the β2 subunit of nicotinic receptors (β2 -/-mice) using a social task that includes an exploratory phase followed by a social interaction phase to promote both novelty exploration and social investigations [START_REF] Granon | Executive and social behaviors under nicotinic receptor regulation[END_REF][START_REF] Avale | Prefrontal nicotinic receptors control novel social interaction between mice[END_REF][START_REF] De Chaumont | Computerized video analysis of social interactions in mice[END_REF]Nosjean et al. 2015Nosjean et al. , 2018)). We showed that acute stress (Nosjean et al. 2015) and that social interaction modulated by the presence of β2-nAChRs, played a buffering role on stress-induced brain activation (Nosjean et al. 2018).

In the latter study, we quantified the regional expression of the protein c-fos to reflect brain activation. C-fos protein expression not only depends on the intensity and the duration of the stimulus, but also on the time-course of the protein translation [START_REF] Honkaniemi | Colocalization of fos-and glucocorticoid receptor-like immunoreactivities in the rat amygdaloid complex after immobilization stress[END_REF]Chowdury et al. 2000;[START_REF] Viau | Hypophysiotropic neurons of the paraventricular nucleus respond in spatially, temporally, and phenotypically differentiated manners to acute vs. repeated restraint stress: rapid publication[END_REF]Muigg et al. 2009; see also [START_REF] Senba | Stress-induced expression of immediate early genes in the brain and peripheral organs of the rat[END_REF][START_REF] Kovács | c-Fos as a transcription factor: a stressful (re)view from a functional map[END_REF][START_REF] Kovács | Measurement of immediate-early gene activation-c-fos and beyond[END_REF]; as reviews). In our previous experiments, the protein expression was measured 90 min after the beginning of stress exposure, i.e., immediately at the end of the social interaction, a delay which falls within the period of peak production of the protein (see [START_REF] Morgan | Mapping patterns of c-fos expression in the central nervous system after seizure[END_REF]Curran 1989, Hoffman et al. 1993;[START_REF] Hughes | Induction of immediate-early genes and the control of neurotransmitter-regulated gene expression within the nervous system[END_REF]Kovacs 1998Kovacs , 2008;;[START_REF] Bisler | Expression of c-Fos, ICER, Krox-24 and JunB in the whisker-to barrel pathway of rats: time course of induction upon whisker stimulation by tactile exploration of an enriched environment[END_REF][START_REF] Guzowski | Mapping behaviorally relevant neural circuits with immediate-early gene expression[END_REF]. It was therefore surprising that only an 8-minute social interaction induced significant and specific brain activation, as a very limited time was left for c-fos protein expression. Thus, in the present study, we question whether PFC and amygdala activity evolve with time by adding a 90 min delay after the social task to allow maximal expression of the protein. To this end, B6 and β2 -/-mice were sacrificed 90 min after either stress alone or after stress followed by the social task. Non-stressed mice were also sacrificed 90 min after the social task.

Materials and Methods

Part of the data used here (all data referring to "short delay") were previously published (Nosjean et al. 2018) as they assess the immediate effect of stress combined or not with a social task on brain activity. As the purpose of the current paper was to compare the effect of a long delay after stress with or without social interactions, to that of a short delay, we included this set of data in our statistical comparisons, in addition to corresponding control data (Fig. 1). This was made possible by the fact that our experiments were conducted in the very same environment and in order to reduce the total number of animals.

Animals

Experiments were conducted with male B6 mice (n = 124) and mice lacking the β2 subunit of the nAchRs (β2 -/-, n = 50). At their arrival to animal facilities, 10-11 weeks old mice were group-housed (four mice/cage; food and water ad libitum, room temperature 20-22°C) under a 12/12h light/dark cycle (light on at 7.30 am). Animals were purchased from Charles Rivers Laboratories (L'Arbresle Cedex, France). β2 -/-mice were generated from a 129/Sv embryonic stem cell line as previously described [START_REF] Picciotto | Abnormal avoidance learning in mice lacking functional high-affinity nicotine receptor in the brain[END_REF] and backcrossed onto the B6 strain for 20 generations. All experiments were carried out to reduce the discomfort of mice in accordance with the EU Directive 2010/63/EU, Decree N 2013-118 of February 1st, 2013, and the French National Committee (87/848).

Experimental procedures (Figure 1)

All experiments were performed from 9.00 a.m. to 2.00 p.m. with 14-25 weeks old mice and conducted for both B6 and β2 -/-mice.

After acclimatization to the experimental room, animals were either immediately sacrificed (Control mice, Cnt, n = 9 B6, n = 6 β2 -/-mice), submitted to an acute stress (S mice), or submitted to acute stress or not and a social task (SESI-mice and ESI-mice). Stress was realized by placing the mouse for 45 minutes in a Falcon® tube opened at the end to permit breathing. Social task was realized by placing an Isolated Host mouse (IH, either stressed or not) and a Social Visitor mouse (SV, never stressed) together for the first time in a novel environment. The IH mouse, previously isolated for 3-4 weeks, was allowed to explore the novel environment for 30 min (E) before gently introducing an unfamiliar SV mouse in the box (always a B6 socially housed mouse). SV mouse was approximately of same weight and age than IH mouse. Social interactions (SI) between mice lasted 8 min. They were recorded for further analysis using Mice Profiler software [START_REF] De Chaumont | Computerized video analysis of social interactions in mice[END_REF].

Mice submitted to stress alone were sacrificed 90 min after the beginning of stress (short delay protocol, S-Short, n = 6 B6, n = 7 β2 -/-mice, published data, Nosjean et al. 2018) or 90 min after its end (long delay protocol, S-Long, n = 7 B6, n = 7 β2 -/-mice, current data). Stressed mice subjected to exploration and social interaction were sacrificed 90 min after the beginning of stress (short delay protocol, SESI-Short, n = 8 B6, n = 8 β2 -/-mice, published data, Nosjean et al. 2018) or 90 min after the end of the social interaction (long delay protocol, SESI-Long, n = 10 B6, n = 7 β2 -/-mice, current data). Non-stressed mice were also sacrificed 90 min after the end of the social interaction (long delay protocol, ESI-Long, n = 8 B6 and n = 8 β2 -/-mice, current data).

Immunohistochemical procedures and quantification

General immunohistochemical procedures and quantification were previously described in details (Nosjean et al. 2018). Briefly, after deep anesthesia (Dolethal 2ml / 3ml Nacl, i.p. 0.1ml / 10g) and intracardiac perfusion of fresh ice cold 4% paraformaldehyde in phosphate buffer saline (PBS), cryoprotected IH brains (20% sucrose in PBS overnight, 4°C°) were cut as serial free-floating coronal sections (40 µm) using a freezing microtome. Rabbit polyclonal anti-c-fos (c-fos (ab-5), 1:8000 dilution, Calbiochem #PC38), biotinylated goat anti-rabbit IgGs secondary antibody (1:200, Vector BA-1000), avidin-biotin complex (1:200 dilution, Vectastain Elite PK 6100) and diaminobenzidine tetrahydrochloride were sequentially used to reveal c-fos protein.

Brain images were acquired at x10 with a Sony DFW-X700 digital camera (Sony Co., Tokyo, Japan) coupled to Olympus BX60 light microscope (Olympus Optical Co., Hamburg, Germany). C-fos positive cells per square micrometer were quantified using icy.bioimageanalysis.org after delineating each region of interest (ROI) on the picture. C-fos positive cells were mapped in all brain structures in front of the corpus callosum from the frontal cortex (defined here for simplicity reasons as the prefrontal cortex, PFC, from bregma +3.08 to +1.54, [START_REF] Paxinos | The mouse brain in stereotaxic coordinates[END_REF], and in the basolateral nucleus of the amygdala (BLA, from bregma -0.58 to -1.58 mm).

In the PFC, for structures extending over more than one rostro-caudal level (i.e., PrL, Cg1, M2, M1, LO, VO and MO, 7 areas), we pooled quantifications obtained from its rostral to its caudal extent (Global PFC). We also analyzed c-fos protein expression by level in the PFC, i.e., in the rostral (from bregma 3.08 to 2.58 mm), median (from bregma 2.46 to 2.10 mm), and caudal parts (from bregma 1.98 to 1.54 mm) of the PFC. For each mouse and each selected brain level, at least 2-3 sections were bilaterally analyzed (i.e., about more than 100 ROIs par mouse) and then averaged for each animal.

Statistical analysis

C-fos protein expression was quantified in the global PFC and in its 24 sub-areas as well as in the BLA of the amygdala. Results were expressed as means ± SEM. Values were considered statistically significant for p ≤ 0.05. Experiments were analyzed in 2 sets. In the first one, comparisons were performed on 10 independent groups, i.e., for both genotypes, in control mice (no stress and no behavioral procedure), in stressed mice (S mice) and in stressed mice submitted to the social task (SESI mice) in which c-fos protein was detected either 90 min after the beginning of the experiment, i.e., after the beginning of the stress (S-Short and SESI-Short mice), or 90 min after the end of the experiment, i.e., after the end of the stress or the social interaction (S-Long and SESI-Long mice). In the second set of experiments, comparisons were performed on 4 independent groups, i.e., for both genotypes, in non-stress (ESI-Long) and stressed (SESI-Long) animals. Multiple comparisons between groups were performed with Kruskal-Wallis one-way ANOVA on Ranks using Sigmaplot 12.0. When normality passed, data were subsequently analyzed with the Holm-Sidak method test and when it did not, with Mann-Whitney U-tests. Correlations between the 25 studied brain areas were performed using Spearman test and a Bonferroni correction was applied for multiple comparisons. Statistically threshold was thus set at p ≤ 0.002.

Results

Effect of time, stress and social interaction on brain activity

In a first series of experiments, we dissected the time effect on brain c-fos protein expression in B6 and β2 -/-mice subjected to acute stress alone or to acute stress followed by social interaction task (Fig. 1).

Effect of time on stress alone (Figure 2A-C, Supplementary Table 1a-b)

Acute stress in B6 mice (S-Short vs Cnt) induced a significant increase of c-fos protein expression in the PrL due to specific activation of the rPrL, in the Cg1 (mCg1 and cCg1), in the M2 (mM2 and cM2), in cM1 and in the BLA. Thus, at short time, acute stress induced brain activation all along the PFC, in particular in the median wall (PrL, Cg1), in the associative and motor areas (FrA, M1, M2) as well as in the BLA. In 2 -/-mice, acute stress followed by a 2 -/-mice compared to their respective controls. Limbic structures such as Prl, Cg1 and the caudal motor cortices (primary and secondary) and BLA were hyper-activated by stress in B6 mice but not in 2 -/-mice while other PFC areas (i.e., mPrL and orbital rLO and rVO areas) and BLA were hypo-activated by stress in 2 -/-mice.

Long delay protocol showed no change in PFC c-fos protein levels, both in stressed B6 and 2 -/-mice compared to their respective controls (S-Long vs Cnt), when considering the entire PFC or its different levels. In contrast, an increase and a decrease of c-fos protein expression remained detected in the BLA respectively in B6 and 2 -/-mice (for both genotypes, H = 36.143, df = 9, p < 0.001; MW: p = 0.001). Thus, time normalized the stress-induced c-fos protein expression in the PFC in both genotypes, but was not sufficient to restore that of the BLA.

Finally, comparisons between short and long delay protocols (S-Long vs S-Short)

showed marginal hypo-activations taking placed only at some levels of the rostral axis: a diminution of c-fos protein expression was observed in B6 mCg1 area (H = 48.526, df = 9, p < 0.001; MW: p = 0.022) and in 2 -/-rFrA (H = 57.449, df = 9, p < 0.001; MW: p = 0.026). No change was detected in B6 BLA (H = 36.143, df = 9, p < 0.001; MW: p = 1.000) while a decrease in 2 -/-BLA was evidenced (H = 36.143, df = 9, p < 0.001; MW: p = 0.007). Such results suggest that time has little if any effect on stress-induced c-fos expression in the PFC, while it influenced the BLA activity, only in 2 -/-mice. This genotype difference discards a simple and non-specific delay effect of cfos protein detection with time. Effect of time on stress followed by social task (Figure 2A-E, Supplementary Table 2a andb) The influence of time was also assessed on social interaction in stressed animals (SESI-Short vs Cnt, SESI-Long vs Cnt, SESI-Long vs SESI-Short).

We observed that B6 mice submitted to acute stress followed by the social task (SESI-Short vs Cnt) showed increased c-fos protein expression in all prefrontal areas (0.03 ≤ ps < 0.001) except in the mVO, the mMO and the cLO (Nosjean et al. 2018). Likewise, 2 -/-animals displayed increased activity compared to their controls (0.011 ≤ ps < 0.001), except in the MO when considering the PFC areas in all their extent and in the rMO, rDLO, mAI, mMO, cAID, cAIV and cLO along the PFC rostro-caudal axis (Nosjean et al. 2018). For both genotypes, hyperactivity was observed in the BLA (H = 36.143, df = 9, ps < 0.001; MW: ps < 0.001). Thus, even if social interaction occurs shortly before sacrifice, i.e., with little time for c-fos expression to change, it produced massive brain activation in stressed mice of both genotypes.

Long delay protocol induced no change in c-fos protein levels when looking at the entire PFC in stressed B6 mice submitted to the social task compared to their own controls (SESI-Long vs Cnt). Along the rostro-caudal axis, it provoked hypo-activity in rFrA (H = 57.449, df = 9, p < 0.001; MW: p = 0.002) and over-activity in mPrL (H = 43.911, df = 9, p < 0.001; MW: p = 0.037), mCg1 (H = 48.526, df = 9, p < 0.001; MW: p =0.045) and BLA (H = 36.143, df = 9, p < 0.001; MW: p = 0.030). In contrast, in 2 -/-mice (SESI-Long vs Cnt), long delay triggered a drop of c-fos protein expression only in LO considering the PFC as a whole (H = 52.467, df = 9, p < 0.001; MW: p = 0.035) likely due to decreases in the rLO (H = 53.706, df = 9, p < 0.001; MW: p = 0.005) and mLO (H = 53.706, df = 9, p < 0.001; MW: p = 0.014).

Other PFC areas also showed hypo-activity: rFrA (H = 57.499, df = 9, p < 0.001; MW: p = 0.002), rVO (H = 46.088, df = 9, p < 0.001; MW: p = 0.005), mPrL (H = 43.911, df = 9, p < F o r P e e r R e v i e w 11 0.001; MW: p = 0.005) and in BLA (H = 36.143, df = 9, p < 0.001; MW: p = 0.001). Thereby, adding a delay between the social interaction and the mice sacrifice drastically changed its impact on brain c-fos protein expression: it diminished the extent of the PFC activation in B6 mice and reversed its impact in 2 -/-mice in which only hypo-activations were detected. Thus, time buffered the stress-social interaction induced c-fos protein activation.

In line with previous work (Nosjean et al. 2018) after a short delay (SESI-Short vs S-Short), social task in stressed mice compared to stress alone triggered over-activations in all rostral areas and only in part in medial and caudal PFC areas (mM1, cM1, rMO, mPrL, cSI and cDI) in B6 mice while in 2 -/-mice only over-activations were detected in the PFC (except in the cLO) and in the BLA. Thus, social task in stressed mice induced hyper-activation at short term, in particular in 2 -/-mice.

After long delay (SESI-Long vs S-Long), social behavior in stressed mice induced no statistical effect for any analyzed PFC area in B6 and 2 -/-mice apart from a hypo-activation in rFrA for both genotypes (H = 57.449, df = 9, p < 0.001; MW: p = 0.002 in B6 mice, p = 0.038 in 2 -/-mice). In the BLA, c-fos protein level dropped in B6 (H = 36.143, df = 9, p < 0.001; MW: p = 0.002) but remained unchanged in 2 -/-mice (H = 36.143, df = 9, p < 0.001; MW: p = 0.535). Thus, the long delay that allowed a maximal expression of c-fos protein revealed a strong buffering role of social interactions on stress-induced prefrontal activation.

Finally, the long delay protocol (SESI-Long vs SESI-Short) provoked hypo-activations in all brain studied areas in both in B6 and 2 -/-mice, except for B6 mice in mVO (F (9,65) = 8.243, p < 0.001; HS: p = 0.614), mMO (F (9,65) = 6.193, p < 0.001; HS: p = 0.294) and cLO (F (9,65) = 3.414, p < 0.001; HS: p = 0.874). Hence, despite some exceptions, the delay before sacrifice induced hypo-activations in the PFC and in the BLA in both genotypes.

Genotype and time effects (Figure 3A-B, Supplementary Table 4a-b)

Comparisons between B6 and 2 -/-mice (2 -/-vs B6 mice) were done for control mice (Cnt), stress alone with a short (S-Short) or a long (S-Long) delay of c-fos protein expression, and stress followed by the social task with a short (SESI-Short) or long delay (SESI-Long).

No statistical differences for any analyzed area were detected between B6 and 2 -/-control mice when considering the PFC or the BLA.

With the short delay following stress alone significant decreases of the protein expression were observed in all PFC areas taken in all their extent in 2 -/-mice compared to B6 mice except in MO (S-Short, 0.029 ≤ ps < 0.001). Hypo-activations were specifically due to diminutions of the protein expression at different levels of the rostro-caudal axis, i.e., in the rLO, rVO, mPrL cPrL mCg1, cCg1, mM2, cM2 and in the BLA which is in concordance with previous data (Nosjean et al. 2018). Hence, in 2 -/-mice, a short delay mostly reduced the protein expression in the orbital structures of the rostral PFC as well as in the medio-caudal wall of the PFC, and in the amygdala, suggesting an important role of 2-nAChRs in these areas.

Stress followed by a long delay in 2 -/-mice compared to B6 mice (S-Long) significantly decreased the activity of PrL (F (9,65) = 16.553, p < 0.001; HS: p = 0.049) and Cg1 (F (9,65) = 15.582, p < 0.001; HS: p = 0.048), likely because of effect on the rPrL (F (9,65) = 15.010, p < 0.001; HS: p = 0.006) and on the cCg1 (F (9,65) = 14.671, p < 0.001; HS: p = 0.041) as other regions remained unaffected. Diminutions in the rostral PFC were also detected, specifically in the rFrA (H = 57.449, df = 9, p < 0.001; MW: p = 0.038) and in the rLO (H = 53.706, df = 9, p < 0.001; MW: p = 0.038). In addition, BLA was hypo-activated (H = 36.143, df = 9, p < 0.001; MW: p < 0.001). Thus, long delay significantly extended hypo-activation in the rostral PFC and in the BLA of 2 -/-mice. Short delay protocol in stressed 2 -/-mice interacting socially compared to their corresponding B6 mice (SESI-Short) didn't show any BLA (H = 36.143, df = 9, p < 0.001; MW: p = 0.574) nor PFC alterations except in rFrA (H = 57.449, df = 9, p < 0.001; MW: p < 0.001). Thereby, the major genotype effect in stressed 2 -/-mice submitted to social task is a restoration of c-fos protein expression in the PFC and in the BLA.

In contrast, at long delay after stress and social task (SESI-Long) there was significant hypo-activations in 2 -/-mice compared to B6 mice in the PrL (F (9,65) = 16.553, p < 0.001; HS: HS: p = 0.016) and in the cCg1 (F (9,65) = 14.671, p < 0.001; HS: p = 0.001). Hence, long delay amplified hypo-activation all along the PFC and in the BLA in 2 -/-mice. As this was not the case in B6 mice, we can rule out a general timing effect of c-fos protein detection. Notably, the genotype effect triggered by any behavioral manipulation (stress, social behavior, at short or long delay) was mainly reflected by hypo-activation of the PFC and of the BLA, but never by hyper-activations. In B6 control mice, 17 positive correlations were detected between PFC sub-areas (0.00199 ≤ ps ≤ 0.0000002), mainly between lateral and medial areas all along the rostro-caudal axis. Stress alone totally disconnected PFC sub-areas activity in B6 mice at short delay (S-Short) but triggered numerous PFC relationships at long delay (41 correlations, S-Long, ps = 0.0000002). These functional connections were evidenced within each level of the PFC as well as between the lateral, medial and latero-medial areas. Only two PFC sub-areas (mVO and cCg1) established functional connectivity with the BLA. B6 mice having a social interaction after stress showed additional correlations at short delay (SESI-Short, 8 correlations; ps = 0.0000002) but drastically disconnected PFC sub-areas at long delay (SESI-Long, 6 correlations; ps = 0.0000002). Only cPrL and cIL remained steadily correlated whatever the delay, when social interaction followed stress.

p = 0.
No significant correlations were found in 2 -/-control animals while stress alone triggered some correlations between PFC areas after short delay, mostly between medial PFC areas (S-Short, 11 positive and 1 negative (bold arrow, ps = 0.0000002). After long delay, stress massively extended functional connectivity within the PFC of 2 -/-mice (S-Long, 82 significant correlations, ps = 0.0000002). Compared to stress alone, correlations following social interaction in stressed 2 -/-animals increased after short delay (SESI-Short, 21 correlations, 0.00178 ≤ ps ≤ 0.0000002), and drastically diminished after long delay (SESI-Long, 17 correlations, ps = 0.0000002). Only cCg1 and cPrL remained steadily correlated whatever the delay, when social interaction followed stress in 2 -/-mice. With time, a very limited number Overall, whatever the experimental procedure, major correlations within PFC sub-areas markedly differed between B6 and 2 -/-mice, while the BLA activity remained mostly independent of the PFC activity. With the long delay stress alone drastically triggered plethora of functional interconnections between PFC sub-areas, while having a social interaction drastically disconnected them in both genotypes. Thus, major changes in the functional connectivity and organization of cortical networks were detected after extended delay. 1,6, Supplementary Tables 5a-b)?

Does stress affect brain activity after a long delay (Figures

In a second series of experiments, we also evaluated the impact of time in stressed and nonstressed mice (SESI-Long and ESI-Long, respectively).

With time, stress triggered hypo-activations in all PFC areas when considering the entire PFC or its different levels both in B6 and in β2 -/-mice interacting socially (0.022 ≤ ps < 0.001 for B6 mice and ps ≤ 0.001 for β2 -/-mice) and in BLA, specifically in β2 -/-mice (F (3,32) = 31.413, p < 0.001; HS: p = 0.002). Indeed, stress had no effect on BLA in B6 mice, suggesting a different emotional value of stress in the two genotypes.

We also assessed the genotype effect in stressed and non-stressed mice interacting socially for a long delay (β2 -/-vs B6 mice). In non-stress condition, when looking at the entire PFC, brain activation didn't differ between the two genotypes except for significant increased activity in M1 (F (3,32) = 34.044, p < 0.001; HS: p = 0.006) and decreased activity in the BLA (F (3,32) = 31.413, p < 0.001; HS: p < 0.001) in β2 -/-mice. Looking at the different levels of the significant only in cM1 (F (3,32) = 37.173, p < 0.001; HS: p = 0.018). Therefore, as compared to baseline levels, the coupling of stress and social interaction relies on functional β2-nAChRs, specifically regarding the medial wall of the PFC and the BLA, in which significant hypoactivations emerged.

We also identified statistical correlations between the different PFC areas taken by levels and the BLA.

In non-stress B6 mice (ESI-Long), 14 positive correlations were detected between PFC sub-areas, mainly between lateral ones along the rostro-caudal axis (0.00178 ≤ ps ≤ 0.0000002).

Stress drastically reduced such functional relationships (SESI-Long, 6 correlations, 0.0000002 ≤ ps ≤ 0.00178). In non-stress 2 -/-mice (ESI-Long), significant correlations were mainly reported in the latero-medial areas at each level of the rostro-caudal axis (19 correlations, 0.00178 ≤ ps ≤ 0.0000002). Correlations were totally different from those identified in B6 mice, except relationships between mM1-cS1 and cCg1-cPrL areas. After stress (SESI-Long, 17 correlations, ps = 0.0000002), correlations between lateral and latero-medial areas were reduced, while those between medial areas appeared.

Overall, at long term the coupling of stress and social interaction drastically changed functional connectivity within the PFC. Although abundant in all brain areas, β2-nAChRs appear particularly important for functional connectivity within prefrontal sub-areas, and for its plasticity and reorganization after stress.

Discussion

The effects of stress exposure depend on many factors such as nature, intensity, duration of the stressor, age, sex and history of the individual. Behavioral and physiological adjustments to stress engage different systems as the neuroendocrine, autonomic, immune, cardiovascular and metabolic systems as well as brain release of different neurotransmitters, neuromodulators and neuropeptides (see [START_REF] Westerink | Brain microdialysis and its application for the study of animal behaviour[END_REF][START_REF] Mcewen | Protective and damaging effects of stress mediators central role of the brain[END_REF][START_REF] Radley | Noradrenergic innervation of the dorsal medial prefrontal cortex modulates hypothalamo-pituitary-adrenal responses to acute emotional stress[END_REF][START_REF] Arnsten | Stress signalling pathways that impair prefrontal cortex structure and function[END_REF][START_REF] Lupien | Effects of stress throughout the lifespan on the brain, behaviour and cognition[END_REF][START_REF] Ossewaarde | Stressinduced reduction in reward-related prefrontal cortex function[END_REF][START_REF] Mcewen | Stress effects on neuronal structure: hippocampus, amygdala, and prefrontal cortex[END_REF], and many brain structures such as the hypothalamus, the hippocampus, the ventral tegmental area, the nucleus accumbens, the PFC and the amygdala, among others (see [START_REF] Cacioppo | Social neuroscience: autonomic, neuroendocrine, and immune responses to stress[END_REF][START_REF] Herman | Neurocircuitry of stress: central control of the hypothalamopituitary-adrenocortical axis[END_REF][START_REF] Buijs | The integration of stress by the hypothalamus, amygdala and prefrontal cortex: balance between the autonomic nervous system and the neuroendocrine system[END_REF][START_REF] Pacák | Stressor specificity of central neuroendocrine responses: implications for stress-related disorders[END_REF][START_REF] De Kloet | Stress and the brain: from adaptation to disease[END_REF][START_REF] Arnsten | Stress signalling pathways that impair prefrontal cortex structure and function[END_REF][START_REF] Lupien | Effects of stress throughout the lifespan on the brain, behaviour and cognition[END_REF][START_REF] Mora | Stress, neurotransmitters, corticosterone and body-brain integration[END_REF][START_REF] Beery | Stress, social behavior, and resilience: insights from rodents[END_REF][START_REF] Mcklveen | The medial prefrontal cortex: coordinator of autonomic, neuroendocrine and behavioural responses to stress[END_REF][START_REF] Paul | Interaction of basal forebrain cholinergic neurons with the glucocorticoid system in stress regulation and cognitive impairment[END_REF]Mc Ewen et al. 2016;Zhang et al. 2018). Brain c-fos protein expression depends on the intensity and the duration of the stimulus and on its acute or chronic character. In the literature acute restraint stress duration has been reported to be either of 30 min [START_REF] Yokoyama | Regional expressions of Fos-like immunoreactivity in rat cerebral cortex after stress; restraint and intraperitoneal lipopolysaccharide[END_REF][START_REF] Chowdhury | Induction and adaptation of Fos expression in the rat brain by two types of acute restraint stress[END_REF][START_REF] Viau | Hypophysiotropic neurons of the paraventricular nucleus respond in spatially, temporally, and phenotypically differentiated manners to acute vs. repeated restraint stress: rapid publication[END_REF][START_REF] Patel | Inhibition of restraint stress-induced neural and behavioural activation by endogenous cannabinoid signaling[END_REF][START_REF] Radley | Noradrenergic innervation of the dorsal medial prefrontal cortex modulates hypothalamo-pituitary-adrenal responses to acute emotional stress[END_REF][START_REF] Goebel | Restraint stress activates nesfatin-1immunoreactive brain nuclei in rats[END_REF][START_REF] Zavala | Female responses to acute and repeated restraint stress differ from those in males[END_REF], 60 min [START_REF] Stone | Effect of locus coeruleus lesion on c-fos expression in the cerebral cortex caused by yohimbine injection or stress[END_REF][START_REF] Yokoyama | Regional expressions of Fos-like immunoreactivity in rat cerebral cortex after stress; restraint and intraperitoneal lipopolysaccharide[END_REF][START_REF] Chowdhury | Induction and adaptation of Fos expression in the rat brain by two types of acute restraint stress[END_REF], two hours [START_REF] Honkaniemi | Colocalization of fos-and glucocorticoid receptor-like immunoreactivities in the rat amygdaloid complex after immobilization stress[END_REF][START_REF] Chowdhury | Induction and adaptation of Fos expression in the rat brain by two types of acute restraint stress[END_REF][START_REF] O'mahony | Restraint stress-induced brain activation patterns in two strains of mice differing in their anxiety behaviour[END_REF] or more (2 at 8 hours; [START_REF] Honkaniemi | Colocalization of fos-and glucocorticoid receptor-like immunoreactivities in the rat amygdaloid complex after immobilization stress[END_REF][START_REF] Chowdhury | Induction and adaptation of Fos expression in the rat brain by two types of acute restraint stress[END_REF][START_REF] Roske | Effect of chronic intermittent immobilization stress on Fos-like immunoreactivity in rat brain and adrenal medulla[END_REF]. The period between the end of the stimulus and the sacrifice of animals for c-fos protein detection also differs: for various stress durations, the delay before sacrifice can be just after stress exposure (Yokohama et al. 1999;[START_REF] Chowdhury | Induction and adaptation of Fos expression in the rat brain by two types of acute restraint stress[END_REF][START_REF] Viau | Hypophysiotropic neurons of the paraventricular nucleus respond in spatially, temporally, and phenotypically differentiated manners to acute vs. repeated restraint stress: rapid publication[END_REF][START_REF] Patel | Inhibition of restraint stress-induced neural and behavioural activation by endogenous cannabinoid signaling[END_REF][START_REF] Reznikov | Activation of phenotypically distinct neuronal subpopulations in the anterior subdivision of the rat basolateral amygdala following acute and repeated stress[END_REF][START_REF] Ito | Green odor" inhalation by rats down-regulates stress-induced increases in Fos expression in stress-related forebrain regions[END_REF]), of 30 min (Yokohama et al. 1999;[START_REF] Viau | Hypophysiotropic neurons of the paraventricular nucleus respond in spatially, temporally, and phenotypically differentiated manners to acute vs. repeated restraint stress: rapid publication[END_REF][START_REF] Patel | Inhibition of restraint stress-induced neural and behavioural activation by endogenous cannabinoid signaling[END_REF], of 1 hour (Yokohama et al. 1999;[START_REF] Viau | Hypophysiotropic neurons of the paraventricular nucleus respond in spatially, temporally, and phenotypically differentiated manners to acute vs. repeated restraint stress: rapid publication[END_REF], of 2 hours [START_REF] Stone | Effect of locus coeruleus lesion on c-fos expression in the cerebral cortex caused by yohimbine injection or stress[END_REF][START_REF] Radley | Regional differentiation of the medial prefrontal cortex in regulating adaptive responses to acute emotional stress[END_REF][START_REF] Radley | Noradrenergic innervation of the dorsal medial prefrontal cortex modulates hypothalamo-pituitary-adrenal responses to acute emotional stress[END_REF][START_REF] O'mahony | Restraint stress-induced brain activation patterns in two strains of mice differing in their anxiety behaviour[END_REF][START_REF] Zavala | Female responses to acute and repeated restraint stress differ from those in males[END_REF], or of several hours [START_REF] Chowdhury | Induction and adaptation of Fos expression in the rat brain by two types of acute restraint stress[END_REF][START_REF] Viau | Hypophysiotropic neurons of the paraventricular nucleus respond in spatially, temporally, and phenotypically differentiated manners to acute vs. repeated restraint stress: rapid publication[END_REF]. Here, with an acute and fix stress duration of 45 min, we addressed the question of impact on brain activity of 1) a time laps between stress coupled or not with a social task, and sacrifice 2) a long delay between social interaction and sacrifice, in stress and no stress conditions, in order to address the plasticity of PFC-BLA networks. We could wonder whether using c-fos is a suitable method to capture the effect of time and its buffering role, as c-fos protein expression varies with time and, as described above, decreases with time. If our results were only due to the time-course of the c-fos expression, we could expect brain activation and brain connectivity to reach control levels after a certain time.

However, our results demonstrate that between short and long delays, we observed either no effect, hyper-activation or hypo-activation, depending on the behavior and of the lack of 2 nAChRs. Similarly, when considering functional connectivity, there was no linear modification with time, thus discarding a simple effect of time-course of the protein expression.

Effect of time, stress, social interaction on brain activity.

First, we analyzed PFC and BLA c-fos protein expression at two time points, i.e., 90 min after the beginning or the end of stress exposure, coupled or not to a social task. Such timing was chosen in accordance with works showing a peak production of the protein between 60-120 minutes [START_REF] Morgan | Mapping patterns of c-fos expression in the central nervous system after seizure[END_REF]Morgan and Curran 1989;[START_REF] Hoffman | c-Fos and related immediate early gene products as markers of activity in neuroendocrine systems[END_REF]Kovacs 1998Kovacs , 2008;;[START_REF] Zangenehpour | Differential induction and decay curves of c-fos and zif268 revealed through dual activity maps[END_REF][START_REF] Guzowski | Mapping behaviorally relevant neural circuits with immediate-early gene expression[END_REF]. We chose to focus on the PFC and on the amygdala because of their key role in mediating the emotional responses to stress and social behavior outcomes [START_REF] Heidbreder | The medial prefrontal cortex in the rat: evidence for a dorso-ventral distinction based upon functional and anatomical characteristics[END_REF][START_REF] Mcewen | Protective and damaging effects of stress mediators central role of the brain[END_REF][START_REF] Mcewen | Central effects of stress hormones in health and disease: Understanding the protective and damaging effects of stress and stress mediators[END_REF][START_REF] Lupien | Effects of stress throughout the lifespan on the brain, behaviour and cognition[END_REF][START_REF] Arnsten | Stress signalling pathways that impair prefrontal cortex structure and function[END_REF][START_REF] Avale | Prefrontal nicotinic receptors control novel social interaction between mice[END_REF][START_REF] Mora | Stress, neurotransmitters, corticosterone and body-brain integration[END_REF]Nosjean et al. 2018). Furthermore, we questioned the role of the cholinergic system using mice lacking nicotinic 2-receptor subunit (2-nAChRs), since brain cholinergic transmission was shown to nAChRs are ubiquitously expressed in the brain, notably in the PFC and the BLA [START_REF] Hogg | Nicotinic acetylcholine receptors: from structure to brain function[END_REF][START_REF] Fowler | Subtypes of nicotinic acetylcholine receptors in nicotine reward, dependence, and withdrawal: Evidence from genetically modified mice[END_REF][START_REF] Hurst | Nicotinic acetylcholine receptors: From basic science to therapeutics[END_REF][START_REF] Mineur | Multiple nicotinic acetylcholine receptor subtypes in the mouse amygdala regulate affective behaviors and response to social stress[END_REF]Zhang et al. 2018;[START_REF] Mineur | The role of acetylcholine in negative encoding bias: Too much of a good thing?[END_REF]) and we previously showed that their viral re-expression specifically in the prelimbic area of the PFC was necessary and sufficient to restore social interaction in 2 -/-mice [START_REF] Avale | Prefrontal nicotinic receptors control novel social interaction between mice[END_REF].

Effect of time on stress alone

At short-term, stress impacted differentially the PFC and the BLA in both genotypes compared to their own control: it activated the BLA and some PFC areas all along the rostro-caudal axis in B6 mice, and hypo-activated the BLA and some other PFC sub-regions in 2 -/-mice. Thus, the BLA and the PFC reacted in opposite way depending on the presence of the 2 nAChRs that play a buffer role in such stress processes. At long term, stress also impacted distinctly each genotype's PFC and BLA reactivity. Indeed, compared to controls, time fully buffers the stressinduced PFC hyper-activation in B6 mice but not the PFC hypo-activation in 2 -/-mice. It had no effect on BLA activity in B6 mice and reinforced the BLA hypoactivation in 2 -/-mice, suggesting that the cholinergic system plays a role on PFC and promotes BLA reactivity.

Interestingly, comparison between short and long delay after stress showed little or no effects in the PFC and the BLA. Thus, the stress impacts fade with time in all PFC areas but was maintained in the BLA. Both stress and time differentially modulate the PFC and BLA activities depending on the presence of 2 nAChRs (Fig. 7).

Despite strong reciprocal anatomical connections between PFC sub-regions [START_REF] Van Eden | Functional neuroanatomy of the prefrontal cortex: autonomic interactions[END_REF][START_REF] Gabbott | Areal and synaptic interconnectivity of prelimbic (area 32), infralimbic (area 25) and insular cortices in the rat[END_REF][START_REF] Heidbreder | The medial prefrontal cortex in the rat: evidence for a dorso-ventral distinction based upon functional and anatomical characteristics[END_REF][START_REF] Dalgleish | The emotional brain[END_REF][START_REF] Vertes | Differential projections of the infralimbic and prelimbic cortex in the rat[END_REF][START_REF] Vertes | Interactions among the medial prefrontal cortex, hippocampus and midline thalamus in emotional and cognitive processing in the rat[END_REF][START_REF] Varela | Insular cortex mediates approach and avoidance responses to social affective stimuli[END_REF], stress in B6 mice completely deconstructs all functional links between PFC sub-areas at short time and triggers multiple PFC relationships at long term suggesting that functional connectivity is an important marker of stress adaptation that should be taken into account in brain imaging studies. By contrast, in 2 -/-mice stress triggers some functional connectivity at short term and plethora at long term. These data evidence that both stress and time participate in network reconfiguration, likely involving 2 nAChRs modulation in the PFC. Analyzing correlation of activity between brain regions using c-fos expression provides an index of functional connectivity [START_REF] Lesburguères | Early tagging of cortical networks is required for the formation of enduring associative memory[END_REF][START_REF] Cohen | Differential stress induced c-Fos expression and identification of region-specific miRNA-mRNA networks in the dorsal raphe and amygdala of high-responder/lowresponder rats[END_REF][START_REF] Varela | Insular cortex mediates approach and avoidance responses to social affective stimuli[END_REF], particularly as we quantified here c-fos expression in numerous prefrontal regions of interest shown to be part of an important social network [START_REF] Varela | Insular cortex mediates approach and avoidance responses to social affective stimuli[END_REF].

Frontal areas established dense reciprocal connections with the BLA [START_REF] Mcdonald | Organization of amygdaloid projections to the prefrontal cortex and associated striatum in the rat[END_REF][START_REF] Mcdonald | Cortical pathways to the mammalian amygdala[END_REF][START_REF] Bacon | Amygdala input to medial prefrontal cortex (mPFC) in the rat: a light and electron microscope study[END_REF][START_REF] Gray | Functional and anatomical relationships among the amygdala, basal forebrain, ventral striatum, and cortex. An integrative discussion[END_REF][START_REF] Vertes | Differential projections of the infralimbic and prelimbic cortex in the rat[END_REF][START_REF] Gabbott | Prefrontal cortex in the rat: projections to subcortical autonomic, motor, and limbic centers[END_REF][START_REF] Hoover | Anatomical analysis of afferent projections to the medial prefrontal cortex in the rat[END_REF]. However, like observed by others in social conditions (Rogers-Carter et al. 2018), we didn't observe functional relationships between the BLA and the PFC sub-regions. Such functional connection significantly emerged only when stress was followed by a long delay, and only in B6 mice, suggesting a role of 2 nAChRs in this PFC-BLA plasticity. Other authors reported that despite anatomical connections some areas (OFC, PrL, hippocampus) may not be activated together, depending on the behavioral contexts [START_REF] Lesburguères | Early tagging of cortical networks is required for the formation of enduring associative memory[END_REF][START_REF] Varela | Insular cortex mediates approach and avoidance responses to social affective stimuli[END_REF][START_REF] Liu | Altered functional connectivityof the amygdala and its subregions in typhoon-related post-traumatic stress disorder[END_REF]. Therefore, we can hypothesize that BLA and PFC may predominantly not act in synergy, except in stress condition after long delay. This synergy could be supported by the fact that stress promotes the release of monoamines, glutamate and GABA both in the PFC and in the amygdala [START_REF] Imperato | Changes in brain dopamine and acetylcholine release during and following stress are independent of the pituitaryadrenocortical axis[END_REF][START_REF] Tanaka | Noradrenaline release in the rat amygdala is increased by stress: studies with intracerebral microdialysis[END_REF][START_REF] Arnsten | Catecholamine regulation of the prefrontal cortex[END_REF][START_REF] Finlay | The effects of stress on central dopaminergic neurons: possible clinical implications[END_REF][START_REF] Feenstra | Dopamine and noradrenaline release in the prefrontal cortex in relation to unconditioned and conditioned stress and reward[END_REF][START_REF] Morilak | Role of brain norepinephrine in the behavioral response to stress[END_REF][START_REF] Moghaddam | Divergent plasticity of prefrontal cortex networks[END_REF]; see also [START_REF] Arnsten | Neurobiology of executive functions: catecholamine influences on prefrontal cortical functions[END_REF][START_REF] Mora | Stress, neurotransmitters, corticosterone and body-brain integration[END_REF]. Acetylcholine, however, plays a distinct role in stress effect. Indeed, stress promotes the release of Ach in the PFC [START_REF] Mark | Inescapable stress enhances extracellular acetylcholine in the rat hippocampus and prefrontal cortex but not the nucleus accumbens or amygdala[END_REF] [START_REF] Pepeu | The acetylcholine, GABA, glutamate triangle in the rat forebrain[END_REF][START_REF] Mora | Stress, neurotransmitters, corticosterone and body-brain integration[END_REF]Ofek and Soreq 2013) but not in the amygdala [START_REF] Mark | Inescapable stress enhances extracellular acetylcholine in the rat hippocampus and prefrontal cortex but not the nucleus accumbens or amygdala[END_REF]. We previously showed that 2 -/-mice exhibit higher levels of monoamines and of Ach than B6 mice in the prelimbic area [START_REF] Coura | Nonaggressive and adapted social cognition is controlled by the interplay between noradrenergic and nicotinic receptor mechanisms in the prefrontal cortex[END_REF], and show altered PFC excitatory/inhibitory balance [START_REF] Pittaras | Neuronal nicotinic receptors are crucial for tuning of E/I balance in prelimbic cortex and for decision-making processes[END_REF]). The differential impact in both strains on stress-induced brain activity/plasticity could be linked to differences in levels of various neurotransmitters, leading to distinct PFC-BLA reactivity to stress.

Effect of time on stress followed by social task

The strong brain c-fos over-expression associated to social interaction occurring in our case 30 minutes after stress in our short-term condition is independent of the time necessary for the maximal protein translation. Brain activation was also seen by others when protein expression was measured just after the end of a stressful experiment [START_REF] Chowdhury | Induction and adaptation of Fos expression in the rat brain by two types of acute restraint stress[END_REF][START_REF] Viau | Hypophysiotropic neurons of the paraventricular nucleus respond in spatially, temporally, and phenotypically differentiated manners to acute vs. repeated restraint stress: rapid publication[END_REF][START_REF] Patel | Inhibition of restraint stress-induced neural and behavioural activation by endogenous cannabinoid signaling[END_REF][START_REF] Reznikov | Activation of phenotypically distinct neuronal subpopulations in the anterior subdivision of the rat basolateral amygdala following acute and repeated stress[END_REF][START_REF] Ito | Green odor" inhalation by rats down-regulates stress-induced increases in Fos expression in stress-related forebrain regions[END_REF]. Moreover, such effects on PFC and BLA activity are specifically due to the social interaction since novelty exploration without social behavior after stress induced only marginal activation of the PFC in B6 mice and no activation in 2 -/-mice (Nosjean et al. 2018). When increasing the delay after social interaction the extent of the PFC hyper-activation was nearly abolished, thus reaching normal levels in B6 mice. PFC activation was, by contrast, completely reversed in 2 -/-mice, in which only hypo-activations were detected. Likewise, only hypo-activations were detected in the entire PFC and in the BLA in both genotypes when comparisons were performed between short and long delays, or in mice submitted to the social task without stress (see Fig. 7). Our data therefore indicates that time alone is not sufficient to restore normal brain activity and that the opportunity to have a social contact after stress promotes brain plasticity.

Stress followed by social task after a long delay differentially engaged PFC and BLA activity in all mice. This is in accordance with the literature assigning a major role of the PFC Grace 2019). We previously showed that acute stress triggers aggressiveness in B6 and β2 -/- mice, even if not all individuals were affected (Nosjean et al. 2015). After a short delay, social interaction restores a few functional links between PFC areas in stressed B6 mice and increases functional connectivity in stressed 2 -/-mice. Increasing the delay after stress coupled to social task drastically reduced the number of functional relationships in both genotypes. Taking together, our data show that functional PFC-BLA connectivity is not only modulated by behavior but also by time and 2 nAChRs activity. Social interaction, known to involve the cholinergic system, and time may together synergistically down-regulate the impact of acute stress and participate in resilience modulation, thus extending the works reviewed by [START_REF] Beery | Stress, social behavior, and resilience: insights from rodents[END_REF].

Does stress affect brain activity after a long delay?

With time, stress coupled with social task hypo-activated all PFC areas in both genotypes while in non-stress condition, restoration to basal level requires β2-nAChRs. Likewise, β2-nAChRs appeared very important for the BLA activity, whatever the emotional state (Fig. 7). Moreover, at long term, coupling acute stress and social task drastically changed the functional connectivity within the PFC but had no effect on the BLA. Thus, β2-nAChRs appear particularly important for functional connectivity within prefrontal sub-areas, and for its plasticity and reorganization after stress. Then, not stressed mice were immediately sacrificed for brain c-fos protein detection (control mice, Cnt). Mice submitted to stress alone (S) or stress and social task (SESI, i.e., stress (S) followed by exploration (E) and social interaction (SI)) were sacrificed either 90 min after the beginning of the stress procedure (Short delay, solid lines) or 90 min after the end of the stress or of the social interaction (Long delay, dotted lines) to allow an identical induction time of cfos protein between S and SESI groups. ESI mice, i.e., not stress mice submitted to exploration and social interaction were sacrificed 90 min after the end of the social interaction (Long delay, dotted lines). IH mouse submitted to the social task was placed in the experimental box for a 30 min novelty exploration period (E) before to introduce a SV mouse in the box during 8 min to study mice social interactions (SI).

Fig. 2. Stress, social task and time effects on c-fos protein expression. A-B:

Quantification of c-fos protein in the PFC and in the BLA in B6 (A) and β2 -/-(B) mice subjected to no stress and no behavior (Cnt), to acute stress alone (S mice) and to acute stress followed by social task (SESI mice). Mice were sacrificed either 90 min after the beginning of the stress (S-Short and SESI-Short) or 90 min after the end of the stress or of the social interaction (S-Long and SESI-Long). Histograms are means ± SEM. Significant differences between groups are reported between bars (0.043 ≤ ps < 0.001 for B6 mice, 0.038 ≤ ps < 0.001 for β2 -/-mice). PFC and amygdalar sub-areas were distinguished using the atlas of [START_REF] Paxinos | The mouse brain in stereotaxic coordinates[END_REF]. Cortical areas were considered either in their extent (from bregma +3.08 to +1.54 mm, Global PFC) or in relation to their rostral (from bregma 3.08 to 2.58 mm, Rostral PFC), median (from bregma 2.46 to 2.10 mm, Median PFC) and caudal (from bregma 1.98 to 1.54 mm, Caudal PFC) location. Letters (r, m, c) preceding the structure's name indicate respectively their rostral, 4a andb for significance of the results). In the representative coronal sections of the rostral, median and caudal PFC and of the amygdala, light gray areas indicate significant decreases of protein expression. Distance to bregma is reported on the right of the coronal sections [START_REF] Paxinos | The mouse brain in stereotaxic coordinates[END_REF]. See Fig. 2 for detailed information on brain subdivisions and brain structures abbreviations, and the number of mice used for each group. the amygdala in stressed and not stressed B6 and 2 -/-mice submitted to the social task and sacrificed 90 min after the end of the social interaction (SESI-Long and ESI-Long, respectively). Histograms are means ± SEM. Significant differences are indicated for each genotype between stressed and not stressed mice (0.002 ≤ ps < 0.001 for B6 mice, ps ≤ 0.001 for 2 -/-mice), and between genotype for not stressed (0.028 ≤ ps < 0.006 for ESI-Long mice) and stressed mice (0.012 ≤ ps < 0.001 for SESI-Long, see supplementary Table 5a and b for significance of the results). In the representative coronal sections of the rostral, median and caudal PFC and of the BLA, light gray areas indicate significant decreases of protein expression and dark gray areas indicate significant increases. Distances to bregma are indicated on the right of the coronal sections [START_REF] Paxinos | The mouse brain in stereotaxic coordinates[END_REF]. See Fig. 2 for detailed information on brain subdivisions and brain structures abbreviations. ESI-Long mice: n = 8 for both genotypes, SESI-Long mice: n = 10 B6 and n = 7 2 -/-mice. the BLA (right) in B6 and 2 -/-mice (solid and dotted lines, respectively). C-fos protein was detected 90 (short) and 180 min (Long) after stress alone and after stress coupled with the social task, and 180 min after the social task without stress. Note that in the PFC, both genotypes exhibited similar profiles with a peak of activity when mice were submitted to stress coupled with the social task and sacrificed 90 min after the beginning of stress or when mice, submitted to the social task without stress were sacificed 180 min after the end of the social interaction.

By contrast, temporal profile of protein expression in the BLA totally differs between genotypes.

F o r P e e r R e v i e w Fig. 1. Experimental procedures. Isolated host B6 or β2-/-mice (IH) and B6 social visitor mice (SV) were kept in experimental room for 1 hour after their transfer from animal facility. Then, not stressed mice were immediately sacrificed for brain c-fos protein detection (control mice, Cnt). Mice submitted to stress alone (S) or stress and social task (SESI, i.e., stress (S) followed by exploration (E) and social interaction (SI)) were sacrificed either 90 min after the beginning of the stress procedure (Short delay, solid lines) or 90 min after the end of the stress or of the social interaction (Long delay, dotted lines) to allow an identical induction time of c-fos protein between S and SESI groups. ESI mice, i.e., not stress mice submitted to exploration and social interaction were sacrificed 90 min after the end of the social interaction (Long delay, dotted lines). IH mouse submitted to the social task was placed in the experimental box for a 30 min novelty exploration period (E) before to introduce a SV mouse in the box during 8 min to study mice social interactions (SI). FrA: frontal association cortex; IL: infralimbic cortex; LO: lateral orbital cortex; M1: primary motor cortex; M2: secondary motor cortex; MO: medial orbital cortex; PrL: prelimbic cortex; S1: primary somatosensory cortex; VO: ventral orbital cortex. C-E: Schematic illustrations of c-fos protein immunoreactivity in representative coronal sections of the rostral, median and caudal PFC in B6 and β2-/-mice. Significant increases or decreases of protein expression are indicated by respectively dark and light gray areas (see supplementary Tables 1a andb

  S-Short vs Cnt, Nosjean et al. 2018) decreased c-fos protein expression only in LO (rLO), rVO, mPrL and in the BLA. Thus, acute stress triggered opposite effects in B6 and

  013), Cg1 (F (9,65) = 15.582, p < 0.001; HS: p = 0.001), LO (H = 52.467, df = 9, p < 0.001; MW: p < 0.001) and BLA (H = 36.143, df = 9, p < 0.001; MW: p < 0.001). Looking at the different levels of the PFC, decreases of the protein expression were significant in the rFrA (H = 57.449, df = 9, p < 0.001; MW: p = 0.017), rLO (H = 53.706, df = 9, p < 0.001; MW: p = 0.003), rVO (F (9,65) = 46.088, p < 0.001; HS: p < 0.001), in the mPrL (H = 43.911, df = 9, p < 0.001; MW: p= 0.002), mCg1 (H = 48.526, df = 9, p < 0.001; MW: p= 0.002), mM2 (H = 45.939, df = 9, p < 0.001; MW: p = 0.028), mLO (H = 40.675, df = 9, p < 0.001; MW: p= 0.001) and the cIL (F (9,65) = 10.503, p < 0.001; HS: p = 0.002), cPrL (F (9,65) = 11.498, p < 0.001;

  Statistical correlations were calculated to identify putative functional relationships between the different PFC areas and the BLA, i.e., between 25 areas.

  similar in B6 and 2 -/-mice after stress (S-Long, 11 correlations) and after stress coupled to social task (SESI-Long, 2 correlations, Fig.4A and B, dotted arrows).

  of the protein were detected in the median and caudal PFC in β2 -/-mice. Such hyper-activations concerned mCg1 (H = 26.937, df = 3, p < 0.001; MW: p = 0.028), mM1 (H = 24.451, df = 3, p < 0.001; MW: p = 0.010), mAI(F (3,32) = 39.367, p < 0.001; HS: p = 0.028), cM2 (H = 26.504, df = 3, p < 0.001; MW: p = 0.050), cM1 (F (3,32) = 37.173, p < 0.001; HS: p = 0.012), cS1(F (3,32) = 20.380, p < 0.001; HS: p = 0.007), cAIV (H = 24.764, df = 3, p < 0.001; MW: p = 0.015) and cLO (H = 27.992, df = 3, p < 0.001; MW: p = 0.015). Hence, in the nonstress condition the absence of functional β2-nAChRs increased activity in the PFC and decreased it in the BLA. Such over-expressions were especially seen in the median and caudal PFC, mostly laterally.In the stress condition only decreases in activity were detected in β2 -/-mice compared to B6 mice. They manifested in all PFC areas of interest, except in M1 (F (3,32) = 34.044, p < 0.001; HS: p = 0.182), as well as in the BLA (F (3,32) = 31.413, p < 0.001; HS: p < 0.001). Significant hypo-activity in the global orbital areas were associated to decreases at each level of the LO (rLO: H = 25.706, df = 3, p < 0.001; MW: p = 0.003; mLO: F (3,32) = 66.234, p < 0.001; HS: p < 0.001), cLO: H = 27.992, df = 3, p < 0.001; MW: p = 0.005), VO (rVO: H = 26.960, df = 3, p < 0.001; MW: p < 0.001; mVO: H = 26.864, df = 3, p < 0.001; MW: p = 0.002) and MO (rMO: F (3,32) = 36.986, p < 0.001; HS: p = 0.015; mMO: F (3,32) = 47.799, p < 0.001; HS: p < 0.001). Likewise, hypo-activity in Prl, Cg1 and in the motor cortices M2 was due to diminutions in their medial and caudal parts (mPrL: H = 26.487, df = 3, p < 0.001; MW: p = 0.002; mCg1: H = 26.937, df = 3, p < 0.001; MW: p = 0.002; cPrL: F (3,32) = 38.701, p < 0.001; HS: p < 0.001; cCg1: H = 26.937, df = 3, p < 0.001; MW: p = 0.002; mM2: F (3,32) = 31.219, p < 0.001; HS: p = 0.013; cM2: H = 26.504, df = 3, p < 0.001; MW: p = 0.004). Decreased activity in the insular cortices also extended along the rostro-caudal axis (mAI: F (3,32) = 39.367, p < 0.001; HS: p = 0.004; cAID: F (3,32) = 22.630, p < 0.001; HS: p = 0.012 and cAIV: HS = 24.764, df = 3, p < 0.001; MW: p = 0.013). Finally, decreases in c-fos protein expression were

  play a major role in stress induced affective and behavioral responses[START_REF] Robbins | Differential regulation of fronto-executive function by the monoamines and acetylcholine[END_REF] Ofek and Soreq 2013;[START_REF] Mineur | The role of acetylcholine in negative encoding bias: Too much of a good thing?[END_REF] Neves and Grace 2019). The 2-

  executive processes and the one of amygdala in affective and avoidance behaviors (see Mc Ewen 2007; del Arco and Mora 2009; Higley and Picciotto 2014; Neves and

  reveal that time fully buffers the stress-induced PFC and BLA hyperactivation in B6 mice. The lack of β2-nAChRs provoked massive PFC and BLA hypoactivations and modifies the functional connectome within the PFC and the functional PFC-BLA connectivity. Part of the PFC areas studied as the dorsomedial PFC, the rostral anterior cingulate cortex, the anterior and ventral median PFC, the rostral insula and the sensory motor areas are largely involved in the default mode network (DMN) characterized by a deactivation of functional brain activity during various tasks and an activation at rest (see[START_REF] Raichle | A default mode of brain function[END_REF][START_REF] Pallesen | Cognitive and emotional modulation of brain default operation[END_REF][START_REF] Raichle | The brain's default mode network[END_REF][START_REF] Mooneyham | Signal or noise: brain network interactions underlying the experience and training of mindfulness[END_REF][START_REF] Clemens | Alerted default mode: functional connectivity changes in the aftermath of social stress[END_REF][START_REF] Finc | Transition of the functional brain network related to increasing cognitive demands[END_REF]. As illustrated in Fig.6, our social task following stress abolished such functional connectivity in the PFC-BLA network. If further works are needed to fully understand the relationship between brain activity and functional connectivity our results clearly show a time dependent brain plasticity.

  Curran T. 1989. Stimulus-transcription coupling in neurons: role of cellular immediate-early genes. Trends Neurosci. 12:459-462. Muigg P, Scheiber S, Salchner P, Bunck M, Landgraf R, Singewald N. 2009. Differential stressinduced neuronal activation patterns in mouse lines selectively bred for high, normal or low anxiety. PLoS One. 4:e5346. Muramatsu I, Masuoka T, Uwada J, Yoshiki H, Yazama T, Lee KS, Sada K, Nishio M, Ishibashi T, Taniguchi T. 2018. A new aspect of cholinergic transmission in the central nervous system. In: Akaike A, Shimohama S, Misu Y, editors. Nicotinic Acetylcholine Receptor Signaling in Neuroprotection [Internet]. Singapore: Springer; Chapter 3. Negrón-Oyarzo I, Aboitiz F, Fuentealba P. 2016. Impaired functional connectivity in the prefrontal cortex: A mechanism for chronic stress-induced neuropsychiatric disorders. Neural. Plast. 7539065. Neves GA, Grace AA. 2019. α7 nicotinic receptor full agonist reverse basolateral amygdala hyperactivity and attenuation of dopaminergic neuron activity in rats exposed to chronic mild stress. Eur. Neuropsychopharmacol. 29 :1343-1353. Nosjean A, Cressant A, de Chaumont F, Olivo-Marin J-C, Chauveau F, Granon S. 2015. Acute stress in adulthood impoverishes social choices and triggers aggressiveness in preclinical models. Front. Behav. Neurosci. 8:447. Nosjean A, de Chaumont F, Olivo-Marin J-C, Granon S. 2018. Stress-induced brain activation: buffering role of social behavior and neuronal nicotinic receptors. Brain Struct. Funct. 223:4259-4274.Ofek K, Soreq H. 2013. Cholinergic involvement and manipulation approaches in multiple system disorders. Chem.

Fig. 1 .

 1 Fig. 1. Experimental procedures. Isolated host B6 or β2 -/-mice (IH) and B6 social visitor

  level. The BLA was considered as extending from bregma -0.58 to -1.58 mm. AI: agranular insular cortex; AID: agranular insular cortex, dorsal part; AIV: agranular insular cortex, ventral part; BLA: basolateral amygdaloid nucleus, anterior part; Cg1: cingulate cortex, area 1; DI: dysgranular insular cortex; DLO: dorsolateral orbital cortex; FrA: frontal association cortex; IL: infralimbic cortex; LO: lateral orbital cortex; M1: primary motor cortex; M2: secondary motor cortex; MO: medial orbital cortex; PrL: prelimbic cortex; S1: primary somatosensory cortex; VO: ventral orbital cortex. C-E: Schematic illustrations of c-fos protein immunoreactivity in representative coronal sections of the rostral, median and caudal PFC in B6 and β2 -/-mice. Significant increases or decreases of protein expression are indicated by respectively dark and light gray areas (see supplementary Tables 1a and b, 2a and b, 3a and b, for significance of the results). Distance to bregma is pointed on the right of the coronal sections. B6: Cnt, n = 9; S-Short, n = 6; S-Long, n = 7; SESI-Short, n = 8; SESI-Long, n = 10. β2 -/-: Cnt, n = 6; S-Short, n = 7; S-Long, n = 7; SESI-Short, n = 8; SESI-Long, n = 7.

Fig. 3 .

 3 Fig. 3. Genotype effects. β2 -/-mice were compared to B6 mice for each experimental

Fig. 4 .

 4 Fig. 4. Correlations between PFC sub-areas and the BLA in B6 (A) and β2 -/-mice (B)

Fig. 5 .

 5 Fig. 5. Stress and social task effects on long term c-fos protein expression in B6 and 2 -/-

Fig. 6 .

 6 Fig. 6. Correlations between PFC sub-areas and the amygdala in B6 (A) and β2 -/-mice (B)

Fig. 7 .

 7 Fig. 7. Schematic representation of the temporal evolution of c-fos protein expression

Fig. 2 .

 2 Fig.2. Stress, social task and time effects on c-fos protein expression. A-B: Quantification of c-fos protein in the PFC and in the BLA in B6 (A) and β2-/-(B) mice subjected to no stress and no behavior (Cnt), to acute stress alone (S mice) and to acute stress followed by social task (SESI mice). Mice were sacrificed either 90 min after the beginning of the stress (S-Short and SESI-Short) or 90 min after the end of the stress or of the social interaction (S-Long and SESI-Long). Histograms are means ± SEM. Significant differences between groups are reported between bars (0.043 ≤ ps < 0.001 for B6 mice, 0.038 ≤ ps < 0.001 for β2-/mice). PFC and amygdalar sub-areas were distinguished using the atlas of[START_REF] Paxinos | The mouse brain in stereotaxic coordinates[END_REF]. Cortical areas were considered either in their extent (from bregma +3.08 to +1.54 mm, Global PFC) or in relation to their rostral (from bregma 3.08 to 2.58 mm, Rostral PFC), median (from bregma 2.46 to 2.10 mm, Median PFC) and caudal (from bregma 1.98 to 1.54 mm, Caudal PFC) location. Letters (r, m, c) preceding the structure's name indicate respectively their rostral, median or caudal level. The BLA was considered as extending from bregma -0.58 to -1.58 mm. AI: agranular insular cortex; AID: agranular insular cortex, dorsal part; AIV: agranular insular cortex, ventral part; BLA: basolateral amygdaloid nucleus, anterior part; Cg1: cingulate cortex, area 1; DI: dysgranular insular cortex; DLO: dorsolateral orbital cortex;

Fig. 3 .Fig. 4 .Fig. 5 .Fig. 6 .Fig. 7 .

 34567 Fig.3. Genotype effects. β2-/-mice were compared to B6 mice for each experimental procedure, i.e., in control mice (Cnt), after a short and a long delay of c-fos protein expression in mice subjected to stress alone (S-Short and S-Long) and to stress followed by social task (SESI-Short and SESI-Long). Quantification (A) and schematic summary (B) of c-fos protein expression in the PFC and in the BLA. Significant differences are indicated (0.029 ≤ ps ≤ 0.001, see supplementary Tables 4a and b for significance of the results). In the representative coronal sections of the rostral, median and caudal PFC and of the amygdala, light gray areas indicate significant decreases of protein expression. Distance to bregma is reported on the right of the coronal sections[START_REF] Paxinos | The mouse brain in stereotaxic coordinates[END_REF]. See Fig.2for detailed information on brain subdivisions and brain structures abbreviations, and the number of mice used for each group.190x253mm (600 x 600 DPI)

  ;

	F
	o r
	P
	e e r
	R e v
	i e w
	22

Table 1a

 1a

	Supplementary Table 2a Supplementary Table 2b Supplementary Table 3b Supplementary Table 4b Supplementary Table 5b
	Supplementary Table 1b
	Supplementary Table 3a Supplementary Table 4a
		Statistical analysis between Statistical analysis between Statistical analysis between 10 groups Statistical analysis between Statistical analysis between		SESI-Short vs Cnt SESI-Short vs S-Short SESI-Long vs Cnt SESI-Long vs S-Long SESI-Long vs SESI-Short SESI-Short vs Cnt SESI -Long vs Cnt SESI-Long vs SESI-Short B6 β2 -/-B6 β2 -/-β2 -/-vs B6 SESI-Long vs ESI-Long β2 -/-vs B6
	S-Short vs Cnt B6 β2 -/-S-Short vs Cnt B6 β2 -/-p p B6 β2 -/-p p <0.001 <0.001 B6 β2 -/-p p <0.001 0.015 SESI-Short vs S-Short S-Long vs Cnt B6 β2 -/-S-Long vs Cnt B6 β2 -/-p p B6 β2 -/-p p 0.807 0.573 B6 β2 -/-p p 0.967 0.888 SESI-Long vs S-Long S-Long vs S-Short B6 β2 -/-S-Long vs S-Short B6 β2 -/-p p B6 β2 -/-p p <0.001 <0.001 B6 β2 -/-p p <0.001 <0.001 B6 β2 -/-B6 β2 -/-Subsequent p p p Cnt S-Short S-Long SESI-Short SESI-Long B6 β2 -/-ESI-Long SESI-Long p test β2 -/-vs B6 Cnt S-Short S-Long Subsequent Subsequent test Subsequent test HS Subsequent test HS Statistical analysis between Statistical analysis between 10 groups F or H p Statistical analysis between 10 groups F or H p 10 groups F or H p F (9,65) = 16.553 < 0.001 10 groups F or H p rPrL F (9,65) = 15.010 Rostral PFC Global PFC PrL Rostral PFC < 0.001 10 groups F or H p F or H p 10 groups 4 groups Rostral PFC Statistical analysis between 10 groups F or H p test p p p p p F or H p Subsequent p p p p test SESI-Short SESI-Long Subsequent test p p p p rPrL F (9,65) = 15.010 < 0.001 HS 0.010 0.008 0.515 0.979 F or H p Rostral PFC Rostral PFC Subsequent test p p p p p Global PFC rFrA H = 57.449, df = 9 < 0.001 MW <0.001 <0.001 0.002 0.038 rPrL F (9,65) = 15.010 < 0.001 HS 0.976 0.364 0.006 0.179 0.860 rPrL F (3,32) = 16.412 < 0.001 HS <0.001 <0.001 0.703 0.297 Global PFC Subsequent test p p p p p p rPrL F (9,65) = 15.010 < 0.001 HS 0.030 0.949 0.120 0.540 0.959 0.544 Cg1 F (9,65) = 15.582 < 0.001 HS <0.001 <0.001 0.458 0.931 0.008 <0.001 rFrA H = 57.449, df = 9 < 0.001 MW <0.001 0.001 0.002 0.002 <0.001 <0.001 PrL F (9,65) = 16.553 < 0.001 HS 0.085 <0.001 0.934 0.949 rDLO F (9,65) = 9.010 < 0.001 HS <0.001 0.008 0.991 0.998 rFrA H = 57.449, df = 9 < 0.001 MW 0.517 0.731 0.038 <0.001 0.017 rFrA F (3,32) = 43.838 < 0.001 HS <0.001 <0.001 0.094 0.102
	Global PFC rFrA H = 57.449, df = 9 < 0.001 M2 F (9,65) = 18.383 < 0.001 rDLO F (9,65) = 9.010 < 0.001 Cg1 F (9,65) = 15.582 rLO H = 53.706, df = 9 PrL F (9,65) = 16.553 < 0.001 rDLO F (9,65) = 9.010 < 0.001 < 0.001 MW HS HS < 0.001 HS HS rDLO F (3,32) = 34.723 < 0.001 HS	0.022 <0.001 <0.001 HS MW 0.916 1.000 <0.001 0.295 <0.001 0.142 0.700 <0.001 0.003 1.000 <0.001 0.072 0.951 1.000 <0.001 <0.001 0.049 0.978	0.101 0.916 0.597 0.978 0.526 0.527 0.969 0.204	1.000 <0.001 <0.001 0.877 0.456 0.013 0.977 0.075	0.026 <0.001 <0.001
	PrL rDLO F (9,65) = 9.010 F(9,65) = 16.553 M1 F (9,65) = 22.532 rLO H = 53.706, df = 9 < 0.001 < 0.001 < 0.001 < 0.001 M2 F (9,65) = 18.383 rVO H = 46.088, df = 9 Cg1 F (9,65) = 15.582 < 0.001 rLO H = 53.706, df = 9 < 0.001 < 0.001 HS HS HS MW < 0.001 HS MW rLO H = 25.706, df = 3 < 0.001 MW	0.030 1.000 <0.001 <0.001 HS MW 0.937 0.768 <0.001 0.917 1.000 <0.001 <0.001 0.281 <0.001 <0.001 0.001 <0.001 0.763 0.998 0.876 0.775 <0.001 0.002 0.048 0.038	0.796 0.998 0.545 0.005 0.929 0.884 0.937 0.105 0.798	0.718 1.000 <0.001 <0.001 0.891 0.456 0.001 0.003 <0.001 0.937 1.000 <0.001 0.003
	Cg1 M2 M1 LO VO MO Amygdala BLA rLO H = 53.706, df = 9 < 0.001 F(9,65) = 15.582 < 0.001 F(9,65) = 18.383 < 0.001 F(9,65) = 22.532 < 0.001 H = 52.467, df = 9 < 0.001 F(9,65) = 11.497 < 0.001 F(9,65) = 10.622 < 0.001 H = 36.143, df = 9 < 0.001 F o MW HS HS HS HS HS MW r P e e 0.021 0.007 0.219 0.112 0.404 0.993 0.002 r R e 0.951 0.988 0.996 0.035 0.625 0.992 0.008 v i e w MW 0.316 0.005 0.751 0.946 0.889 0.997 0.832 0.997 1.000 0.001 rVO H = 46.088, df = 9 < 0.001 MW 0.216 0.035 0.832 rMO F (9,65) = 11.936 < 0.001 HS 0.995 0.980 0.997 Median PFC mPrL H = 43.911, df = 9 < 0.001 MW 0.087 0.022 0.397 mCg1 H = 48.526, df = 9 < 0.001 MW 0.029 0.295 0.244 mM2 H = 45.939, df = 9 < 0.001 MW 0.022 0.731 0.169 mM1 F (9,65) = 19.382 < 0.001 HS 0.788 0.977 0.901 mAI F (9,65) = 7.448 < 0.001 HS 0.942 0.966 1.000 mLO H = 40.675, df =9 < 0.001 MW 0.953 0.051 0.138 mVO F (9,65) = 8.243 < 0.001 HS 0.939 0.575 0.997 mMO F (9,65) = 6.193 < 0.001 HS 0.998 0.968 0.993 F o r P e e r R e v i e w LO H = 52.467, df = 9 < 0.001 MW <0.001 <0.001 0.967 VO F (9,65) = 11.497 < 0.001 HS 0.002 0.011 1.000 MO F (9,65) = 10.622 < 0.001 HS <0.001 0.052 0.997 Amygdala BLA H = 36.143, df = 9 < 0.001 MW <0.001 <0.001 0.030 F o r P e e r R e v i e w rVO H = 46.088, df = 9 < 0.001 MW <0.001 0.008 0.307 rMO F (9,65) = 11.936 < 0.001 HS <0.001 0.063 0.988 Median PFC mPrL H = 43.911, df = 9 < 0.001 MW 0.008 <0.001 0.037 mCg1 H = 48.526, df = 9 < 0.001 MW 0.008 0.001 0.045 mM2 H = 45.939, df = 9 < 0.001 MW 0.002 0.001 0.838 mM1 F (9,65) = 19.382 < 0.001 HS <0.001 <0.001 0.309 mAI F (9,65) = 7.448 < 0.001 HS 0.007 0.142 1.000 mLO H = 40.675, df =9 < 0.001 MW 0.030 0.001 0.775 mVO F (9,65) = 8.243 < 0.001 HS 0.659 0.027 0.994 mMO F (9,65) = 6.193 < 0.001 HS 0.397 0.208 1.000 Caudal PFC F o r P e e r R e v i e w M1 F (9,65) = 22.532 < 0.001 HS <0.001 <0.001 LO H = 52.467, df = 9 < 0.001 MW 0.081 <0.001 VO F (9,65) = 11.497 < 0.001 HS 0.755 <0.001 MO F (9,65) = 10.622 < 0.001 HS 0.010 0.001 Amygdala BLA H = 36.143, df = 9 < 0.001 MW 1.000 <0.001 F o r P e e r R e v i e w rMO F (9,65) = 11.936 < 0.001 HS <0.001 <0.001 Median PFC mPrL H = 43.911, df = 9 < 0.001 MW 0.043 <0.001 F o r M2 F (9,65) = 18.383 < 0.001 HS 0.915 <0.001 0.291 M1 F (9,65) = 22.532 < 0.001 HS 0.992 0.029 0.997 LO H = 52.467, df = 9 < 0.001 MW 0.953 0.001 0.097 F o r rVO H = 46.088, df = 9 < 0.001 MW 0.517 0.008 0.318 rMO F (9,65) = 11.936 < 0.001 HS 0.997 0.819 0.272 Median PFC F o r F o r rVO H = 26.960, df = 3 < 0.001 MW <0.001 <0.001 rMO F (3,32) = 36.986 < 0.001 HS <0.001 <0.001 F F Median PFC o r o r mCg1 H = 48.526, df = 9 < 0.001 MW 0.142 <0.001 mM2 H = 45.939, df = 9 < 0.001 MW 0.081 <0.001 mM1 F (9,65) = 19.382 < 0.001 HS <0.001 <0.001 mAI F (9,65) = 7.448 < 0.001 HS 0.690 0.001 mLO H = 40.675, df =9 < 0.001 MW 0.228 <0.001 mVO F (9,65) = 8.243 < 0.001 HS 0.992 <0.001 mMO F (9,65) = 6.193 < 0.001 HS 0.551 0.002 Caudal PFC cIL F (9,65) = 10.503 < 0.001 HS 0.658 <0.001 P e e r R e v i e w VO F (9,65) = 11.497 < 0.001 HS 1.000 0.002 0.779 MO F (9,65) = 10.622 < 0.001 HS 0.949 0.702 0.430 P mPrL H = 43.911, df = 9 < 0.001 MW 0.377 0.008 0.165 P P mPrL H = 26.487, df = 3 < 0.001 MW <0.001 <0.001 P P Amygdala BLA H = 36.143, df = 9 < 0.001 MW 0.680 0.001 <0.001 e e r mCg1 H = 48.526, df = 9 < 0.001 MW 0.377 0.002 0.097 mM2 H = 45.939, df = 9 < 0.001 MW 0.596 0.001 0.165 e e r e e r mCg1 H = 26.937, df = 3 < 0.001 MW 0.002 <0.001 mM2 F (3,32) = 31.219 < 0.001 HS <0.001 <0.001 e e r e e r R e v i e w mM1 F (9,65) = 19.382 < 0.001 HS 0.992 0.211 0.996 mAI F (9,65) = 7.448 < 0.001 HS 0.973 0.204 0.980 mLO H = 40.675, df =9 < 0.001 MW 0.517 0.138 0.535 mVO F (9,65) = 8.243 < 0.001 HS 0.954 0.051 0.911 mMO F (9,65) = 6.193 < 0.001 HS 1.000 0.973 0.995 Caudal PFC R e v i e w R e v i e w mM1 H = 24.451, df = 3 < 0.001 MW 0.001 <0.001 mAI F (3,32) = 39.367 < 0.001 HS <0.001 <0.001 mLO F (3,32) = 66.234 < 0.001 HS <0.001 <0.001 mVO H = 26.864, df = 3 < 0.001 MW <0.001 <0.001 R e v R e v mMO F (3,32) = 47.799 < 0.001 HS <0.001 <0.001 Caudal PFC i e w i e w	0.954 0.878 0.995 0.234 0.937 0.675 0.001 0.138 0.445 0.666 0.234 0.534 1.000 0.933 0.965 0.101 0.666 0.619 0.035 0.312 0.576 0.001 0.005 0.584 0.005 0.234 0.295 0.781 0.687 0.014 0.173 0.565 0.997 0.591 1.000 1.000 0.002 0.993 0.464 0.893 0.602 0.382 0.195 0.584 0.721 0.809 0.188 0.130 0.993 0.999 0.107 0.997 0.992 0.953 0.999 0.838 1.000 0.442 0.574 0.878 0.382 0.028 0.718 0.360 0.997 0.645 0.643 0.998 0.010 0.028 0.176 0.161 0.257	0.407 0.339 0.059 0.138 0.482 0.996 1.000 0.534 0.366 0.960 0.534 0.022 0.181 0.092 0.894 0.181 0.795 0.992 <0.001 0.001 <0.001 <0.001 <0.001 <0.001 0.001 <0.001 <0.001 <0.001 0.003 0.011 0.614 0.294 0.972 0.456 0.908 1.000 0.535 0.998 0.093 0.976 <0.001 <0.001 <0.001 0.940 0.989 0.822 0.710 0.998 0.988 0.007 0.456 0.318 0.994 0.902 1.000 0.620 0.994 0.997 0.805 0.999 0.999 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.587 <0.001 0.015 0.902 0.710 0.318 0.694 0.996 0.318 0.989 1.000 0.942 0.075 0.002 0.002 0.348 0.002 0.028 0.002 0.013 <0.001 0.990 0.646 0.001 0.102 0.456 0.807 0.004 <0.001 0.002 <0.001
	Caudal PFC cIL cIL	F (9,65) = 10.503 cPrL F (9,65) = 11.498 < 0.001 F (9,65) = 10.503 < 0.001 cIL F (3,32) = 43.271 < 0.001 < 0.001 HS HS HS	<0.001 HS 0.854 0.004	0.008 0.567 0.092 <0.001 0.785 <0.001 0.861	0.506 0.934 0.983 0.114	0.038 0.954 0.002 <0.001	<0.001
	cIL cPrL F (9,65) = 11.498 F (9,65) = 10.503 cCg1 F (9,65) = 14.671 < 0.001 < 0.001 cPrL F (9,65) = 11.498 < 0.001 cPrL F (3,32) = 38.701 < 0.001 < 0.001 HS HS HS HS	0.278 <0.001 HS 0.941 0.007	0.954 0.002 0.819 0.018 <0.001 0.927 0.559 <0.001 0.678	0.967 0.921 0.984 0.971 0.115	0.562 0.023 0.867 0.016 <0.001	0.986 <0.001
	cPrL F (9,65) = 11.498 cCg1 F (9,65) = 14.671 cM2 F (9,65) = 14.671 < 0.001 < 0.001 cCg1 F (9,65) = 14.671 < 0.001 cCg1 H = 24.215, df = 3 < 0.001 < 0.001 HS HS HS MW	0.135 <0.001 HS 0.981 0.004 <0.001 0.986 0.611 <0.001 <0.001 0.983 0.232 <0.001 0.041	0.963 0.983 0.972 0.987 0.195	0.559 0.036 0.970 0.001 0.002	0.983 <0.001
	cCg1 F (9,65) = 14.671 cM2 F (9,65) = 14.671 cM1 F (9,65) = 19.667 < 0.001 < 0.001 cM2 F (9,65) = 14.671 < 0.001 cM2 H = 26.504, df = 3 < 0.001 < 0.001 HS HS HS MW	0.012 <0.001 HS 0.952 <0.001 0.962 <0.001 0.012 <0.001 <0.001 0.778 0.982 <0.001 0.164	0.978 0.917 0.999 0.820 0.050	0.575 <0.001 0.920 0.058 0.004	0.967 <0.001
	cM2 F (9,65) = 14.671 cM1 F (9,65) = 19.667 cS1 F (9,65) = 24.159 < 0.001 < 0.001 cM1 F (9,65) = 19.667 < 0.001 cM1 F (3,32) = 37.173 < 0.001 < 0.001 HS HS HS HS	0.003 <0.001 HS 1.000 <0.001 0.877 <0.001 <0.001 0.059 <0.001 0.915 0.995 <0.001 0.997	0.977 0.480 0.875 0.977 0.012	0.173 <0.001 0.477 0.688 0.018	0.918 <0.001
	cM1 F (9,65) = 19.667 cS1 F (9,65) = 24.159 cDI F (9,65) = 11.341 < 0.001 < 0.001 cS1 F (9,65) = 24.159 < 0.001 cS1 F (3,32) = 20.380 < 0.001 < 0.001 HS HS HS HS	0.029 <0.001 HS 0.999 0.005 <0.001 1.000 <0.001 0.995 <0.001 1.000 1.000 <0.001 0.358	1.000 0.998 1.000 0.993 0.007	0.202 <0.001 0.931 0.999 0.722	1.000 <0.001
	cS1 F (9,65) = 24.159 cDI F (9,65) = 11.341 cAID F (9,65) = 8.254 < 0.001 < 0.001 cDI F (9,65) = 11.341 < 0.001 cDI F (3,32) = 12.906 < 0.001 < 0.001 HS HS HS HS	0.994 <0.001 HS 1.000 0.022	1.000 0.004 0.062 1.000 <0.001 1.000 1.000 0.014 0.966	0.976 0.999 0.999 0.996 0.059	0.967 <0.001 0.803 1.000 0.716	0.992 <0.001
	cDI F (9,65) = 11.341 cAID F (9,65) = 8.254 cAIV F (9,65) = 8.319 < 0.001 < 0.001 cAID F (9,65) = 8.254 < 0.001 cAID F (3,32) = 22.630 < 0.001 < 0.001 HS HS HS HS	0.993 <0.001 HS 0.993 0.009	1.000 0.145 0.122 0.941 <0.001 0.969 0.539 <0.001 0.999	1.000 0.539 0.995 0.978 0.063	0.996 <0.001 0.995 0.276 0.012	0.995 <0.001
	cAID F (9,65) = 8.254 cAIV F (9,65) = 8.319 cLO F (9,65) = 3.414 < 0.001 < 0.001 cAIV F (9,65) = 8.319 < 0.001 cAIV H = 24.764, df = 3 < 0.001 < 0.001 HS HS HS MW	0.657 <0.001 HS 0.995 0.005	0.999 0.081 0.998 0.696 <0.001 0.998 0.961 0.599 0.996	0.999 0.439 0.999 0.990 0.015	0.657 0.021 0.604 0.101 0.013	0.994 <0.001
	cAIV F (9,65) = 8.319 cLO F (9,65) = 3.414 cLO F (9,65) = 3.414 cLO H = 27.992, df = 3 < 0.001 < 0.001 < 0.001 < 0.001	HS HS	HS MW	0.960 0.985 0.926 <0.001 0.954 0.999 0.985 <0.001 0.998 0.998 0.999	0.967 0.117 1.000 0.015	0.996 0.874 0.124 0.005	0.890 0.004
	cLO F (9,65) = 3.414	< 0.001	HS		0.999	0.993	0.994	0.999	0.992	0.999
										2 3 4 5 6 7 8 10

Acknowledgements

We thank Jonathan Chabout, Claire Dechoux and Julie Brissez for help with some immunohistochemical experiments.

Notes

Funding

This work was supported by the Centre National de la Recherche Scientifique (CNRS, UMR 9197) by the ANR-FLEXNEURIM grant.

Conflict of Interest: None declared. Supplementary Captions to Tables.

Supplementary Table 5a

Supplementary Table 1. Statistical analyses in B6 and 2 -/-mice submitted to acute stress (S) when c-fos protein was detected 90 min after the beginning (short delay, S-Short) or the end of the stress procedure (long delay, S-Long; see Fig. 1). For each genotype, comparisons were done with their respective controls (Cnt) and between S-Long and S-Short protocols. Comparisons were performed on 10 independent groups with Kruskal-Wallis one-way ANOVA on Ranks (p) in the Global PFC and in the amygdala (a), and in the rostral, median and caudal PFC (b). When normality passed, data were subsequently analyzed with the Holm-Sidak method test (F) and when data did not follow a Gaussian distribution we performed Mann-Whitney U-tests (H). p of post-hoc comparisons are indicated (post-hoc). Gray areas indicated significant differences.

Supplementary Table 2. Statistical analyses in B6 and 2 -/-mice submitted to stress followed by exploration and social interaction (SESI) when c-fos protein was detected 90 min after the beginning of stress procedure (short delay, SESI-Short) or 90 min after the end of social interaction (long delay, SESI-Long; see Fig. 1).

For each genotype, comparisons were done with their respective controls (Cnt) and between SESI-Long and SESI-Short protocols. Comparisons were performed on 10 independent groups using Kruskal-Wallis one way ANOVA on Ranks (p) in the Global PFC and in the amygdala (a) and in the rostral, median and caudal PFC (b). See supplementary Table 1 for statistical comparisons. Gray areas indicated significant differences. 3. Statistical analyses in B6 and 2 -/-mice submitted to stress alone (S) and to stress followed by exploration and social interaction (SESI). For each genotype, comparisons were done when c-fos protein was detected 90 min after the beginning of stress (short delay, SESI-Short vs S-Short) and 90 min after the end of stress or social interaction (long delay, SESI-Long vs S-Long; see Fig. 1). Statistical analyses were performed on 10 independent groups using Kruskal-Wallis one-way ANOVA on Ranks (p) in the Global PFC and in the amygdala (a) and in the rostral, median and caudal PFC (b). See supplementary Table 1 for statistical comparisons. Gray areas indicated significant differences. 4. Statistical comparisons between B6 and 2 -/-mice submitted to no stress and no behavior (Control mice, Cnt), to stress alone (S) with c-fos protein detected 90 min after the beginning of stress (short delay, S-Short), 90 min after the end of stress (long delay, S-Long), 90min after the beginning interaction (SESI-Long) (see Fig. 1). Statistical analyses were performed on 10 independent groups using Kruskal-Wallis one way ANOVA on Ranks (p) in the Global PFC and in the amygdala (a) and in the rostral, median and caudal PFC (b). See supplementary Table 1 for statistical comparisons. Gray areas indicated significant differences.

Supplementary Table

Supplementary Table

Supplementary Table 5. Statistical analyses in B6 and 2 -/-mice submitted to exploration and social interaction following or not acute stress (SESI and ESI mice, respectively) when c-fos protein was detected 90 min after the end of the social interaction (long delay, see Fig. 1B). Comparisons were done between SESI-Long and ESI-Long mice for each genotype (middle columns) and between B6 and 2 -/-for each experimental condition (2 -/-versus B6 mice, ESI-Long and SESI-Long, right columns). Comparisons were performed on 4 independent groups with Kruskal-Wallis one-way ANOVA on Ranks (p) in the Global PFC and in the amygdala (a), and in the rostral, median and caudal PFC (b). See supplementary Table 1 for statistical comparisons. Gray areas indicated significant differences.