Inhibition of the medial amygdala disrupts escalated aggression in lactating female mice after repeated exposure to male intruders

Maternal aggression is a crucial adaptive behavior in rodents, protecting offspring from infanticide. While lactating dams readily exhibit this aggression, its intensity varies across the postpartum period and depends on intruder characteristics. Previous research showed that pup-sensitized virgin mice, co-housed with dams and pups, readily care for pups but do not display intruder-directed aggression during a single testing session. This study aimed to determine if repeated testing would induce aggression in these virgins and to investigate the role of the medial amygdala (MeA) in the escalated aggression observed in dams. The MeA is a key brain region integrating vomeronasal, olfactory, and hormonal information, and its disruption has been linked to deficits in various social behaviors, including maternal aggression. The hypothesis was that the MeA plays a crucial role in the expression of maternal aggression, particularly the escalation observed with repeated intruder encounters. Chemosensory signals, particularly pheromones in urine, are thought to be critical in mediating sociosexual behaviors, including maternal aggression. The study hypothesized that repeated exposure to an intruder male would escalate aggression in dams, potentially involving learning and motivational factors, and that MeA inactivation would prevent this escalation.

The literature extensively documents the hormonal and neural control of maternal aggression in rodents. Studies have shown the importance of hormonal changes during pregnancy and lactation, highlighting the role of oxytocin, vasopressin, and prolactin. The vomeronasal and olfactory systems are crucial for processing chemosensory cues, and the MeA plays a central role in integrating these signals with hormonal information. Previous research from the authors demonstrated that pup-sensitized virgins exhibit pup care but not aggression in single testing sessions, suggesting that aggression requires hormonal changes beyond those related to pup care. Studies have shown that experience-dependent increases in aggression occur in male mice and involve the MeA and its projections to the ventromedial hypothalamus (VMH). The use of DREADDs allows for the precise chemogenetic manipulation of neuronal activity, providing a valuable tool to investigate the causal role of MeA in aggression.

The study consisted of two experiments. Experiment 1 compared intruder-directed aggression in lactating dams (n=8) and pup-sensitized virgin females (n=8) across three postpartum days (PPD4-PPD6). Each female was exposed to a different male intruder for 5 minutes daily. Behavioral measures included total duration of attacks, latency to attack, anogenital investigation, and body approach. Experiment 2 investigated the role of the MeA using DREADDs. Female mice (n=35) received stereotaxic injections of DREADD-AAV vectors into the MeA. After a recovery period, they were mated and assigned to two groups. One group received clozapine-N-oxide (CNO) on PPD4 and vehicle on PPD5, and the other received vehicle on PPD4 and CNO on PPD5. Control groups without DREADD injections were also included. Females were then exposed to male intruders for 5 minutes on PPD4 and PPD5. Behavioral measures were the same as in Experiment 1. Immunohistochemistry was used to verify injection sites. Data were analyzed using repeated measures ANOVA and non-parametric tests where appropriate.

Experiment 1 showed a significant increase in the duration and intensity of maternal aggression in dams across testing days. Dams displayed significantly more attacks of longer duration on the last day compared to the first. Pup-sensitized virgins, however, showed negligible aggression even after repeated exposure. Experiment 2 demonstrated that MeA inactivation did not completely block the initial expression of maternal aggression but prevented the expected increase in aggression on the second testing day. Chemogenetic inhibition of the MeA prevented the escalation of aggression observed in the vehicle-treated group. Analysis of latency to attack revealed a shorter latency to attack in the group receiving CNO in PPD4 than PPD5 in the group which received the vehicle in PPD5, supporting the observation that MeA inactivation prevented escalated aggression, but did not prevent innate maternal aggression.

The findings confirm that maternal aggression escalates with repeated testing in dams, possibly due to a combination of learning and increased motivation. The lack of aggression in pup-sensitized virgins, despite pup exposure and repeated testing, suggests that hormonal changes during pregnancy and lactation are necessary for the development of this behavior. The MeA plays a crucial role in experience-dependent aggression escalation. The MeA's role likely involves the integration of chemosensory cues from intruders with hormonal status, influencing the switch from attraction to aggression. The results support a common neural circuit for intermale and maternal aggression, with sexual dimorphism potentially residing in the expression of hormonal receptors within the MeA and its target areas.

This study demonstrates that repeated exposure to male intruders escalates maternal aggression in dams, a phenomenon that depends on the MeA. Pup-sensitized virgins do not develop this aggression, highlighting the importance of hormonal changes during pregnancy and lactation. The MeA integrates chemosensory and hormonal signals to modulate aggression. Future research should focus on specific neuronal populations within the MeA to better understand the mechanisms underlying this experience-dependent aggression increase.

One limitation is the relatively short duration (5 min) of each behavioral test session. Longer testing durations might reveal differences in the behavior of pup-sensitized females. Another limitation is that the use of a different intruder male each day introduces a confound of novel odors and appearance into the repeated exposure. Finally, the study focused on the medial amygdala and future research is required to investigate other brain areas, particularly the VMH, and more generally on the circuits and mechanisms underlying the experience-dependent changes in aggression.