MSEW results in adulthood maladaptive affective behaviors
To assess whether MSEW contributes to the development of depression-like symptoms, we exposed animals to forced swim test (FST) in early age and in adulthood. We evaluated the immobility latency and total immobility time (Fig. 1E-H). Although ethological analyses revealed no effect of stress or sex, there was a main effect of age in both immobility latency and immobility time (p = 0.005, p = 0.047, respectively). This effect was manifested in the pairwise comparison with Bonferroni’s correction which revealed a specific increase in the total immobility time of adult males (p = 0.019). To further validate these results, we measured the number of immobile episodes during the FST (Supplementary Fig. 2D). A main effect of age x sex was found (p = 0.007) and the differences between groups were shown in MSEW males in early age, performing more immobile episodes than CT group (p = 0.018).
To check locomotor activity and anxiety-like behavior, the open field (OF) test was evaluated in adulthood. In particular, the distance travelled (Fig. 1I-J) by the animals and the ratio between the time they spent in the center versus the time they spent in the periphery of the apparatus were measured (TC/TP; Fig. 1K-M).
No statistically significant differences were observed in locomotor activity neither in males nor in females (Fig. 1I-J). However, the TC/TP ratio was decreased in MSEW males (p = 0.021) compared with CT males (Fig. 1L). MSEW males spent less time in the center and more time in the periphery area compared to CT males, an indicator of anxious state (Supplementary Fig. 2F-G). Interestingly, these changes were especially manifested when considering the last 5 min of the test (Fig. 1M, p = 0.027), but not during the first 5 minutes, ruling out the possible novelty effect of the apparatus and suggesting the escalated anxiety of these animals (Supplementary Fig. 2E). As no effect of stress was found in locomotor activity in the OF test (see before), differences in the TC/TP ratio or other behavioral parameters cannot be attributed to locomotor impairments. MSEW females, by contrast, did not show differences in the TC/TP ratio when compared to their CT group (Fig. 1K, M, Supplementary Fig. 2E-G), suggesting that females were more resilient than males to the long-term effects of ELS on anxiety-related behavior.
Taken together, MSEW exposure effectively induces maladaptive affective behaviors in adult mice in a sex-dependent manner.
MSEW triggers more novelty exploration in females but less social interest in males than CT mice
To test social behavior (Fig. 2A-I), we used a three-chamber apparatus (3CH). After determining during a 10-minute habituation period that there was no preference or bias for spending time in one specific side (Supplementary Fig. 2A-B), social behavior (Fig. 2F-G) and novel preference (Fig. 2H-I) were evaluated.
During early age and adulthood, no main effects of stress or sex condition were detected between groups on either sociability (% time exploring the social target) or novel preference (% time exploring the novel target) (Supplementary Table 2). No differences were reported in the % of time exploring the social target in the sociability test in females (Fig. 2F), but MSEW adult males demonstrated to be significantly less sociable than their respective controls (p = 0.011, Fig. 2G). Conversely, MSEW females spent more time exploring the novel mouse than CT females (p = 0.015, Fig. 2H) and MSEW males did not show differences during this novelty preference phase (Fig. 2I).
Altogether, these findings prompt to think that early life adversity could elicit pro-social behavior in adult females (preference for the novelty) while in adult males could trigger non-social behavioral phenotypes.
Aggressive-like behavior and social dominance emerges in early life and persists until adulthood in both sexes when animals are exposed to MSEW
To address the question of whether ELS induces aggressive-like behavior or regulates social dominance in the short and long-term (Fig. 2J-O), we next exposed the animals to the Tube Test (TT).
The TT results during early life demonstrate that MSEW enhanced social dominance in both females and males, as revealed by the dominance score across all rounds (p < 0.001, p < 0.001 respectively). Moreover, in adulthood, these differences were also significant, being higher in MSEW females (p = 0.043) and males (p = 0.002) than their corresponding controls (Fig. 2L-M).
To further evaluate the social rank across rounds, we also considered the differences between groups in each encounter (Supplementary Fig. 1C). Female and male mice exhibited a higher victory rate and higher probability to become dominant in the first encounter, as evidenced by the significant differences in Round 1 both in early age and adulthood (females p = 0.011, males p < 0.001; females p = 0.033, males p = 0.006, respectively). These differences were kept along the rounds and age, excepting the last round in adulthood (Supplementary Table 2). These data indicate that mice are prone to win a social competition in the TT after ELS and are also likely to maintain their social rank along time.
To explore the effects of MSEW in aggression, we also analyzed the aggressive-like behavior in the TT by measuring the average number of pushes that mice carried out during encounters (Fig. 2N-O). Main effects of age and age x stress were found (p < 0.001, p < 0.001). Specifically, MSEW females and MSEW males displayed a higher number of pushes than their respective controls in early age (p = 0.001, p < 0.001; Supplementary Table 2). These effects were also manifested in adulthood since MSEW females and males showed higher number of pushes than controls (p = 0.012, p = 0.008). These results suggest that ELS led to increased aggressiveness during adulthood.
MSEW decreases neuronal density in different nuclei of the amygdala in a sex and age-dependent manner
Importantly, there is a lack of data regarding the effects of ELS on the cytoarchitecture of the amygdala, thus, we investigated the neuronal composition of its different nuclei (Fig. 3). We described the density of three different neuronal subpopulations in the MeA, BLA, and central amygdala (CeA) along different bregma levels (Fig. 3B): (i) mature excitatory neurons (NeuN + GAD67-SST-), (ii) interneurons lacking somatostatin expression (NeuN + GAD67 + SST-) and (iii) somatostatin interneurons (NeuN + GAD67 + SST+) (Fig. 3C-D).
When analyzing how these subpopulations were affected by ELS both after maternal separation and, afterwards, at adulthood we found some interesting results, such as an age-dependent maturation of SST + interneurons in both MeA and BLA (p = 0.002 and p = 0.004, respectively). Interestingly we also found a main effect of stress, showing reduced density of both NeuN + GAD67-SST- and NeuN + GAD67 + SST + neurons in the BLA (p = 0.031 and p < 0.001, respectively) and CeA (p = 0.015 and p = 0.020, respectively) (See Supplementary Table 2).
Although no significant changes in the pairwise comparisons were observed in the MeA (Fig. 3E-G), in the BLA, we found a decrease in the density of SST + interneurons of MSEW females at early age (p = 0.006), and in MSEW-males and MSEW-females at adulthood (p = 0.009 and p = 0.012 respectively) (Fig. 3K). Moreover, a decrease in the density of SST lacking interneurons of stressed adult females (p = 0.018) was reported within the CeA.
Together, these results indicate that SST + interneurons are indeed affected by ELS, and that those alterations can last until adulthood, thus, having the potential to mediate some of the consequences of ELS in adult brains.
MSEW induces low neuronal activation in MeA SST+ interneurons in adult males correlated to negative socio-aggressive outcomes
Then, we proceeded to investigate the direct influence of MSEW on the neuronal activity of these cells within the amygdala. To pursue this idea, we measured the expression of cFos in the different neuronal subpopulations previously defined, immediately after MSEW (P17), to evaluate their implication right after the stress model, and then in adulthood, to study the long-lasting effects of ELS on the basal activity of these subpopulations (Fig. 4A-K).
When analyzing the NeuN + GAD67-SST- neurons, we observed main effects of stress in the MeA and the CeA (p = 0.044 and p = 0.038, respectively). Significant changes in the activity of NeuN + GAD67-SST- neurons were found right after the MSEW protocol, at early age, specifically on females, in the BLA and CeA (p = 0.004 and p = 0.017 respectively; Fig. 4F, I). A similar alteration was found with NeuN + GAD67 + SST- neurons in the MeA (p = 0.020, Fig. 4D), where females also displayed increased c-Fos expression after MSEW.
However, when considering the activation levels of NeuN + GAD67 + SST + neurons, we observe a decrease of c-Fos expression in adult MSEW-males (p = 0.049, MeA), confirming that the activity of these interneurons is affected by ELS (Fig. 4E, lower graph).
To deepen in this hypothesis, we explored the statistical correlation between the c-Fos levels of SST + interneurons in adult animals and their behavior (Fig. 4L). Interestingly, we found a direct correlation between the expression of c-Fos in these interneurons and their sociability (r = 0.553, p = 0.008, Fig. 4M), and in the same line, an inverse correlation with the aggressivity traits (r= -0.592, p = 0.004; Fig. 4N).
These results suggest that early life stress alters the activity of SST + interneurons, especially those of the MeA, and that the level of their activity is a good predictor for traits of social interaction and aggressivity in mice.
In vivo chemogenetic manipulation of MeASST+ interneurons modulates stress-induced aggression behavioral phenotypes
Given our previous findings showing (i) significant correlations between cFos expression in MeASST+ interneurons and socio-aggressive behaviors and, (ii) the decreased activity of these neurons in MSEW-adult males in combination with the former maladaptive traits, we explored whether chemogenetic activation of MeASST+ interneurons could reverse these phenotypes.
Following the same MSEW protocol, SST-Cre male mice were injected with a virally encoded Cre-dependent chemogenetic actuator in the MeA, and then behaviorally tested both pre and post CNO administration.
First, we confirmed that acute CNO treatment significantly increased cFos expression in the infected neurons in a subset of animals (p = 0.0015, Fig. 5E). Then, we analyzed the behavioral traits related to social dominance and aggression in the TT (Fig. 5F-G). Although we did not find any alteration in the social dominance (Fig. 5F), excitingly, we found that hM3Dq activation of MeASST+ neurons promoted a significant decrease in the number of pushes in MSEW-hM3Dq group compared to mCherry group (p = 0.0406, Fig. 5G). These results indicate that the boost of the activity in MeASST+ neurons reflect a pervasive attenuation of aggression. Furthermore, CNO administration did not affect aggressiveness or dominance in animals with AAV-mCherry i.e., animals without DREADDs, which argues against any unspecific effect of the CNO.
Going deeper in these results, we observed the ethological evolution of each animal along the three rounds. No differences were reported in any round relative to dominance score (Supplementary Fig. 2D) and number of pushes/s (Fig. 5H) after MeASST+ neuronal activation indicating that the difference between both groups is the average over the three rounds (p = 0.0295). These results demonstrate that MSEW-mCherry and MSEW-hM3Dq mice have an equal probability to become dominant in the first encounter of the TT, and equal aggressive-traits in each round.
Moreover, no differences were observed pre CNO treatment in dominance or aggression, hence both MSEW groups prior treatment were equally balanced (Supplementary Fig. 2D, Fig. 5H).
To determine whether SST-neuronal activity manipulation in the MeA is sufficient to improve sociability, we exposed the animals to the 3CH test to evaluate the social interest and the novel preference. No significant differences were shown in any of the 3CH phases after normalizing to each preCNO value (Fig. 5I-K).
To better understand the social interaction of those animals in a more naturalistic environment, animals underwent the RI test in their home-cages and behavior was analyzed with advanced annotation and tracking systems (DeepLabCut, SimBA). Regarding social dominance, there were no differences between groups in tail rattling neither in bout counts nor in duration (Fig. 5N-O), supporting the previous results of the dominance score in the TT. Activation of MeASST+ neurons decreased offensive behavior by decreasing the number of attacks (p = 0.0035, Fig. 5P) and its duration (p = 0.0142, Fig. 5Q) compared to the empty-DREADD group. Moreover, we also evaluated the social interaction, and CNO administration did not reduce the number of social bout counts (Fig. 5R). However, the duration of the social interaction was increased in the MSEW-hM3Dq group relative to MSEW-mCherry group (p = 0.0387, Fig. 5S), indicating that the diminution in aggression could reflect a pervasive improvement of social interest.
In summary, MeASST+ neurons play a specific and essential role in the display of male territorial aggression and social interaction. These results suggest that altering specifically the activity of MeASST+ interneurons is a potential target to reduce aggressive behavior.
To establish whether the manipulation of SST + interneurons specifically modulated the offensive-like behavior, we differentiated between this behavior and the defensive-like. Supporting our hypothesis, no differences were found neither in defensive bout counts (Supplementary Fig. 2E) nor defensive duration (Supplementary Fig. 2F).
We additionally analyzed self-grooming, as an indicative of repetitive behavior, and rearing for exploratory behavior. In self-grooming, no differences were reported in bout counts or duration (Supplementary Fig. 2G-H). Regarding rearing, a slightly decrease in bout counts and duration (p = 0.0363 and p = 0.0075, respectively) could be observed in MSEW-mCherry compared to MSEW-hM3Dq group. However, in MSEW-hM3Dq group, mean values of rearing duration and number of rearing bouts were very close to one after normalization to preCNO results meaning almost no effect of DREADD activation in this group (Supplementary Fig. 2I-J).