Since early stress has been implicated in antisocial behaviors 
, we examined whether social exploration was altered by MSUS in F1, F2 and F3 mice. We first assessed the animals' response to their 1st
exposure to a same-sex unfamiliar conspecific (non-threatening juvenile) that normally elicits high levels of social interaction and investigation of the conspecific. F1 MSUS and control males spent a comparable amount of time investigating their conspecific (), however F2 and F3 MSUS males investigated their conspecific significantly less than controls, suggesting social anxiety or social withdrawal (). This trait was specific to males since social investigation was not altered in F2 and F3 MSUS females (). During the 2nd
exposure to the same conspecific, control animals investigated their conspecifics less than during the 1st
exposure, due to habituation resulting from appropriate social recognition memory. However, F1 MSUS males had no reduction, suggesting a deficit in social memory () 
. Likewise, F2 and F3 MSUS females showed no decrease in investigation during 2nd
exposure to a conspecific. The deficit was specific to social memory since F1, F2, and F3 MSUS animals were able to form and retain a memory of olfactory cues not related to conspecifics similarly to controls (). These results suggest a combination of social anxiety and impaired social memory in MSUS mice that is transmitted across generations. The profile of transmission was consistent across the 2nd
generations even though social anxiety was not clearly expressed in F1 MSUS males. Thus, these males appear to act as asymptomatic carriers, transmitting social anxiety to their F2 offspring, which themselves transmit this trait further to F3 offspring. This may result from the fact that F1 animals were exposed to MSUS only postnatally, while F2 offspring carry the effect transmitted from their father through both pre- and postnatal development, and therefore are more affected. The presence of these abnormal behaviors in the F2 and F3 MSUS generation suggests an epigenetic mechanism involving the germline.
Abnormal sociability in F2 and F3 MSUS males.
Abnormal social memory in F1, F2 and F3 MSUS mice.
Early deficits in serotonin signaling via the serotonin (5HT) 1A receptor (5HT1AR) lead to heightened social anxiety in 5HT1AR knockout mice 
, and patients with social anxiety disorder have reduced 5HT1AR binding in the brain 
. We examined whether 5HT1AR binding is altered by MSUS, in particular in the offspring of MSUS males, by using a radioligand with high affinity for the 5HT1AR, 8-OH-DPAT. Binding assays revealed that 8-OH-DPAT binding is reduced in several brain areas important for anxiety, stress and defense reactions in MSUS mice when compared to controls. These brain regions included the lateral, but not dorsal periaqueductal grey (PAG), the dorsal raphe (DR), but not median raphe (MR), CA1 and dentate gyrus (DG), but not CA2/3 subregions of the hippocampus, and several regions of the thalamus (). Regions in the hypothalamus, cortex, and striatum were not affected (). While 8-OH-DPAT binds 5HT1AR selectively, it can also bind other receptors such as the 5HT7R and the alpha1-adrenoreceptor, albeit with much lesser affinity 
. Thus, the reduced binding in F2 MSUS mice, although predominantly resulting from reduced 5HT1AR binding, may also reflect alterations in other receptor pathways.
5HT1AR binding and serotonin level in the brain of MSUS offspring.
Since 5HT1ARs in the DR and MR are autoreceptors that inhibit serotonergic projections to several brain areas 
, we next examined whether serotonin release is increased in areas that receive DR projections in MSUS mice. To investigate this possibility, we measured the level of serotonin in the frontal cortex, a DR target area, and dorsal hippocampus, a MR target area, in F2 MSUS mice by high-performance liquid chromatography (HPLC). Consistent with the hypothesis, we found that the level of serotonin was increased in frontal cortex, but not in dorsal hippocampus, in F2 MSUS mice (), suggesting a functional consequence of reduced 5HT1AR binding in DR. This finding significantly extends previous data demonstrating enhanced serotonin release following uncontrollable and chronic stress, and reduced autoreceptor activation in the DR 
, by newly showing that such functional alterations are persistent and can be transmitted across generations. To confirm that altered serotonergic signaling is also involved in social anxiety and its transmission to following generations in MSUS mice, we assessed the effect of the selective 5HT1AR agonist, 8-hydroxy-2-dipropylaminotetralin (8-OH-DPAT), on social behaviors. Acute administration of 8-OH-DPAT fully reversed the deficit in social interaction in F3 MSUS males, and normalized the time animals spent investigating their conspecific (). This experiment was also performed in F3 females, but no conclusions could be drawn due to social avoidance induced by the injection itself in both control and MSUS mice (results not shown). However, the data in males suggest that similar molecular alterations involving reduced signaling via the 5HT1AR underlie the transmissible social anxiety observed in both F2 and F3 MSUS mice.
Previous work has demonstrated that rodents exposed to chronic stress show stress resilience when later subjected to aversive and stressful conditions 
. We examined whether early stress results in transmissible alterations in response to social conflict by exposing F2 MSUS and control mice to chronic social defeat (SD). SD is a highly aversive form of social conflict that in rodents, impairs behavioral and neuroendocrine responses, and induces depressive symptoms such as anhedonia, strong avoidance of cues associated with defeat, stereotyped behaviors, and decreased exploratory, motivational and socio-sexual behaviors 
. When exposed to chronic SD, control males consumed significantly less sucrose during the two weeks of SD (anhedonia) (), and showed significant avoidance of odor cues from an aggressor when compared to non-SD controls (). In contrast, F2 MSUS males showed no reduction in sucrose consumption when compared to non-SD control or MSUS males, despite exposure to a similar number of attacks (). They also demonstrated significantly less avoidance of odor cues from an aggressor than SD control males ().
Altered response to social defeat in F2 MSUS males.