Full-length ArticleEarly life stress perturbs the maturation of microglia in the developing hippocampus
Introduction
Abnormal hippocampal development in children exposed to abuse or neglect has been reported by several groups (Chugani et al., 2001b, Carrion et al., 2010, Maheu et al., 2010, Garrett et al., 2012, Herringa et al., 2013). Maltreated children show abnormal hippocampal activation that is associated with impaired declarative memory (Chugani et al., 2001a). Adolescents that were abused or neglected have abnormal hippocampal connectivity that is correlated with the severity of their mood and anxiety symptoms (Herringa et al., 2013). Exposure to early life stress also causes hippocampal abnormalities in nonhuman primates (Spinelli et al., 2010, Jackowski et al., 2011) and rodents (Bredy et al., 2003, Poeggel et al., 2003, Champagne et al., 2008, Ivy et al., 2010, Wei et al., 2012, Wei et al., 2015, Wang et al., 2013). For example, repeated maternal separation increases spine density in the hippocampus (Helmeke et al., 2001, Poeggel et al., 2003), and low levels of maternal care, early in life, are associated with reduced synaptic density in the hippocampus and poor spatial learning in adulthood (Liu et al., 2000, Bredy et al., 2003, Champagne et al., 2008).
Unfortunately, most of the animal work to date has focused on the effects of early life stress on the function and structure of the hippocampus in adulthood, with little effort made to elucidate the mechanisms by which stress early in life alters neurodevelopment (Kaffman and Krystal, 2012). To address this issue, we developed a mouse model of early life stress in which pups of the highly stress-responsive strain, Balb/cByj, are exposed to brief daily separation (BDS) during the first three weeks of life in the absence of nesting material (impoverished condition). We showed that exposure to BDS causes prolonged elevation of corticosterone in 14-day old pups and blunted the rapid increase in total DNA and RNA normally seen in the hippocampus during this age (Wei et al., 2010, Wei et al., 2012, Wei et al., 2014). Exposure to BDS was also associated with a robust increase in anxiety-like behavior and significant deficits in several hippocampal-dependent tasks in adult mice (Wei et al., 2010, Wei et al., 2012), consistent with the notion that BDS is a mouse model of early life stress. We have replicated these findings across multiple cohorts of Balb/cByj mice and believe that the absence of nesting material, coupled with daily separation, in this stress-reactive strain are responsible for the robust and deleterious outcomes seen in BDS mice (Wei et al., 2010, Wei et al., 2012, Wei et al., 2015). More recently, we showed that exposure to BDS impairs synaptic maturation and pruning in the hippocampus of 28-day old mice (Wei et al., 2015). Exposure to BDS also reduces the expression of proteins involved in axonal growth and myelination in the hippocampus, alterations that were detected as early as PND14 (Wei et al., 2015).
Microglia, the brain’s innate immune resident cells, play a critical role in guiding many developmental processes that are affected by BDS (Wei et al., 2015). These include synaptic pruning (Paolicelli et al., 2011, Schafer et al., 2012), synaptic maturation (Lenz et al., 2013, Parkhurst et al., 2013), neurogenesis (Butovsky et al., 2006, Wakselman et al., 2008, Cunningham et al., 2013, Ueno et al., 2013), axonal growth (Chamak et al., 1994), and myelination (Miron et al., 2013, Bhatt et al., 2014). In addition, microglia dynamically probe the developing brain and are highly responsive to several mediators of stress such as corticosterone (Wu et al., 2001, Frank et al., 2011), catecholamines (Johnson et al., 2005, McNamee et al., 2010, Wohleb et al., 2011), and corticotropin-releasing hormone (Stevens et al., 2003, Ock et al., 2006, Kritas et al., 2014). Abnormal activation of microglia has been shown to perturb several developmental processes in a manner that persists in adulthood (Schafer et al., 2012, Cunningham et al., 2013, Lenz and McCarthy, 2015). These properties led us to investigate the effects of BDS on microglia function in the developing hippocampus.
Here we show that exposure to BDS increased the density and altered the morphology of microglia in the hippocampus of 14-day old pups, effects that were no longer present at PND28. However, using the NanoString immune panel we found that BDS altered expression of many immune-related genes at both ages. We also showed that during normal hippocampal development, microglia undergo significant changes between PND14 and PND28 that include reduced cell density, decreased ex vivo phagocytic activity, and increased expression of genes involved in inflammation and cell migration. Hippocampal microglia isolated from 28-day old BDS mice showed an increase in phagocytic activity and reduced expression of genes that normally increase across development. Promoter analysis indicated that alteration in the transcriptional activity of PU.1, Creb1, Sp1, and RelA accounted for most of the transcriptional changes seen during normal microglia development and in response to BDS at both PND14 and PND28. These findings suggest that BDS impairs the normal maturation of microglia in the developing hippocampus and provide a possible mechanism to explain at least some of the neurodevelopmental abnormalities seen in the hippocampus of Balb/cByj mice exposed to BDS.
Section snippets
Animals
BALB/cByj mice (Stock # 001026, Jackson Laboratories) were housed in standard Plexiglas cages and kept on a standard 12:12 h light-dark cycle (lights on at 7:00 AM), at constant temperature and humidity (22 °C and 50%), with food provided ad libitum. The Institutional of Animal Care and Use Committee (IACUC) at Yale University approved all studies reported here.
Brief daily maternal separation (BDS)
The BDS procedure was done as described previously (Wei et al., 2010). In brief, visibly pregnant dams were placed individually in
BDS increases anxiety-like behavior in juvenile mice
We have previously shown that exposure to BDS increases anxiety-like behavior in adulthood (Wei et al., 2010, Wei et al., 2012). However, the effects of BDS on these behaviors in juvenile mice have not been examined yet. To address this issue, we tested two anxiety-like behaviors (e.g. open field and elevated plus maze) in juvenile mice that were raised under control or BDS condition (See Fig. 1A for timeline). We focused on male mice because our previous work (Wei et al., 2010, Wei et al., 2012
Discussion
Exposure to BDS recapitulates several clinical findings reported in maltreated children, suggesting good construct validity as a model of childhood adversity. These include: elevated corticosterone levels during the postnatal period, abnormal hippocampal development, and reduced myelination (Wei et al., 2010, Wei et al., 2012, Wei et al., 2014, Wei et al., 2015). In this report we show that the increase in anxiety-like behaviors, previously reported in adult BDS mice (Wei et al., 2010, Wei et
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
We thank Drs. Eric Wohleb and Evelyn Cumberbatch for helpful comments on the manuscript. This work was supported by: NIMH R21MH098181, NIMH R01MH100078, and the Clinical Neuroscience Division of the VA National Center for PTSD, the NIH-NINDS (1R01NS088137) (O.B.), National Multiple Sclerosis Society (5092A1) (O.B.) and Nancy Davis Foundation Faculty Award (O.B.).
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