We found that treating rats with LPS on day 15 of pregnancy reduced social play of male but not of female offspring. Only one other study reports that LPS exposure on day 9.5 of pregnancy reduces play in male offspring; no females were studied, and no effects were found on gross morphology of the brain . We found that, in addition to social play, LPS reduced AVP expression in the BST and MeA, again in male but not in female offspring. Therefore, our findings suggest that the often-made observation that males are more vulnerable to prenatal stress than females [25, 26] can be extended to the neural substrate underlying social behavior in juvenile animals.
Social play as well as AVP expression in the BST and MeA are more prominent in male than in female juveniles [20, 22]. As LPS treatment reduced social play and AVP expression in males only, LPS treatment may have interfered with general mechanisms of sexual differentiation. For example, malnutrition or environmental stress in the last week of pregnancy reduces differentiation of sexual behavior and SDN-POA volume . A stress-linked reduction in fetal activity of testosterone may contribute to this effect [28, 29]. As sexual differentiation of play behavior and AVP expression depends on higher levels of testosterone in males [30–33], LPS may have inhibited masculinizing effects of testosterone. Our data, however, argue against a general effect of LPS on sexual differentiation, as the volume of the SDN-POA was unaffected by LPS treatment.
There are several other possible explanations as to why LPS treatment affected AVP expression in males but not in females in the present study. Humoral factors generated as a result of LPS treatment may have differential access to male and female fetuses. For example, stress early in pregnancy significantly changes the expression of genes implicated in the hypoxic response, cell differentiation, and metabolism in male but not in female placentas . Immune challenges may have similar dimorphic effects on the placenta, thereby possibly differentially affecting the exchange of nutrients, metabolic waste products, and signaling molecules across the placental barrier.
LPS treatment may also have made AVP cells less sensitive to the masculinizing effects of gonadal steroids postnatally. Higher levels of testosterone found in males increase the probability that developing neurons in the BST and MeA commit to a vasopressinergic phenotype [35, 36]. Given that LPS effects were only significant in males, LPS may have interfered with this differentiating step. Interestingly, sexual differentiation of specific brain areas and behaviors uses components of signal transduction pathways that are common to inflammatory processes [37–39]. As AVP cells in the BST are affected by inflammatory processes in adult animals , there may be cross-talk between sexual differentiation and immune signaling pathways during their development as well.
Several lines of evidence suggest that the LPS-induced reduction in play behavior and in AVP mRNA expression in the MeA are causally related. AVP has been implicated in the control of social behavior [6, 41]. Moreover, injecting an AVP receptor antagonist intracerebroventricularly reduces play behavior in males . Furthermore, systemic testosterone can masculinize play behavior as well as AVP expression in the MeA and BST by acting on androgen rather than estrogen receptors [30, 33], and intracranial testosterone implants placed specifically into the amygdala masculinize social play . It is not yet known whether AVP treatment can reverse the reduction in play behavior in LPS Males.
The LPS-induced reduction in AVP mRNA was specific to the BST and MeA, as levels did not change it in the SON, PVN, or SCN. Differences in birth date of AVP neurons may contribute to differences in LPS effects on various AVP-expressing brain regions in this study. AVP neurons in the BST and MeA are born on embryonic days 11 and 12 [42, 43] and therefore all of these cells could be affected by LPS treatment on embryonic day 15. In contrast, SCN cells are born on embryonic days 14–17 , which is, by and large, at or after the LPS treatment given in this paper. Differences in developmental trajectory may also shelter the PVN and SON, as neurons in these nuclei are born on embryonic days 12–18. Thus only a fraction of the cells are born by the time LPS was administered .
BST and MeA cells appear to be responsive to immune challenges in adulthood as well. In adult rats, LPS treatment acutely increases AVP release in the ventral septal area , a projection area of AVP neurons of the BST and MeA , and treatment with the pro-inflammatory cytokine interleukin-1beta, which is released upon exposure to LPS, increases the firing rate of BST and MeA neurons . These effects have been linked to AVP’s role in fever abatement . We propose that LPS treatment early in life may activate these same neurons, thereby permanently changing their impact on physiology and behavior. A relevant example of such programming is found in the administration of LPS early postnatally, which permanently alters the fever response, interestingly, more so in males than in females .
As of yet it is unclear what molecular mechanisms underlie LPS-induced changes in AVP expression and play behavior. Most likely, early immune activation altered the fate of a number of cells, perhaps by changing epigenetic regulation of AVP gene expression. Such long-term changes have been shown for the PVN, where early life stress increases AVP expression while reducing methylation of CpG sites of a chromosomal region that controls AVP expression . Related epigenetic changes might underlie the effects of prenatal immune activation on social behaviors reported here.
In addition to LPS treatment directly affecting fetal development, it may have altered maternal behavior, and thereby development. Several studies suggest that this is not likely. For example, dams injected with the same dose of LPS used in this study, but on embryonic day 15 as well as day 16, did not show changes in parental care . In addition, stress during fetal development changes social behavior and expression of oxytocin mRNA in the PVN of adult male rats, irrespective of whether they were raised by stressed or unstressed dams . However, given the role of maternal care in male sexual differentiation , the possibility remains that changes in maternal behavior may have mediated or masked potential effects of prenatal LPS.