Arie Kaffman

Early-life stress, corpus callosum development, hippocampal volumetrics, and anxious behavior in male nonhuman primates

Male bonnet monkeys (Macaca radiata) were subjected to the variable foraging demand (VFD) early stress paradigm as infants, MRI scans were completed an average of 4 years later, and behavioral assessments of anxiety and ex-vivo corpus callosum (CC) measurements were made when animals were fully matured. VFD rearing was associated with smaller CC size, CC measurements were found to correlate with fearful behavior in adulthood, and ex-vivo CC assessments showed high consistency with earlier MRI measures. Region of interest (ROI) hippocampus and whole brain voxel-based morphometry assessments were also completed and VFD rearing was associated with reduced hippocampus and inferior and middle temporal gyri volumes. The animals were also characterized according to serotonin transporter genotype (5-HTTLPR), and the effect of genotype on imaging parameters was explored. The current findings highlight the importance of future research to better understand the effects of stress on brain development in multiple regions, including the corpus callosum, hippocampus, and other regions involved in emotion processing. Nonhuman primates provide a powerful model to unravel the mechanisms by which early stress and genetic makeup interact to produce long-term changes in brain development, stress reactivity, and risk for psychiatric disorders.

Affiliative Behavior Requires Juvenile, But Not Adult Neurogenesis

The capacity to interact with conspecifics is essential for stable social networks, reproduction, and survival in mammals. In rodents, social exploration and play behavior increase during the juvenile period, suggesting that this timeframe represents an important window for socialization. However, the cellular and molecular mechanisms necessary to support this developmental process have not been elucidated. Neurogenesis during the juvenile period, like that in adults, is mainly confined to the subgranular and subventricular zones. Nevertheless, the levels of neurogenesis are significantly higher during the juvenile period, suggesting unique functions not shared with adult neurogenesis. Here we use a transgenic mouse approach that allows for ablation of neurogenesis during different developmental phases. We find that ablating neurogenesis during either juvenile or adult phases altered anxiety and memory in adult female mice, demonstrating an age-independent function of new neurons for certain behaviors. Blocking neurogenesis during the juvenile period resulted in a profound impairment in the ability of these mice to interact with other adult females or to retrieve pups, without causing gross olfactory deficits. Interestingly, ablating neurogenesis in adult females had no effect on these social behaviors. This work defines a novel role for juvenile neurogenesis in establishing brain circuits necessary for socialization, and demonstrates that juvenile and adult neurogenesis make different contributions to social competency in adult female mice. Additional work is needed to determine whether ablation of juvenile neurogenesis in the subgranular zone and/or the subventricular zone is responsible for the social abnormalities seen after global elimination of juvenile neurogenesis.

Early Life Stress Inhibits Expression of a Novel Innate Immune Pathway in the Developing Hippocampus

Childhood maltreatment represents a major risk factor for the development of numerous childhood psychopathologies that in many cases linger as chronic mental illnesses in adulthood. Exposing rodents or non-human primates to early life stress increases anxiety-like behaviors and impairs cognitive function in adulthood, suggesting that animal models may provide important insights into parallel developmental processes in humans. Using an unbiased genomic screen, we found that expression of lipopolysaccharide binding protein (LBP), a member of the innate immune system, is dramatically decreased in the hippocampus of pups exposed to early life stress. LBP levels peak in the normally developing hippocampus at a period of intense synaptic pruning, during which LBP is colocalized with the synaptic marker PSD95 and is found in close proximity to processes of microglia cells. Expression of LBP declines to low levels seen in adulthood at around postnatal day 30. Importantly, 30-day-old LBP knockout (k.o.) mice show increased spine density and abnormal spine morphology, suggesting that peak levels of LBP during the second and third weeks of life are necessary for normal synaptic pruning in the hippocampus. Finally, LBP k.o. mice show impaired hippocampal-dependent memory and increased anxiety-like behaviors in a manner that resembles that seen in animals exposed to early life stress. These findings describe a novel role for LBP in normal hippocampal development and raise the possibility that at least some of the behavioral sequelae of early life stress are mediated by reduced expression of LBP during a critical period of neurodevelopment.

New Frontiers in Animal Research of Psychiatric Illness

Alterations in neurodevelopment are thought to modify risk of numerous psychiatric disorders, including schizophrenia, autism, ADHD, mood and anxiety disorders, and substance abuse. However, little is known about the cellular and molecular changes that guide these neurodevelopmental changes and how they contribute to mental illness. In this review, we suggest that elucidating this process in humans requires the use of model organisms. Furthermore, we advocate that such translational work should focus on the role that genes and/or environmental factors play in the development of circuits that regulate specific physiological and behavioral outcomes in adulthood. This emphasis on circuit development, as a fundamental unit for understanding behavior, is distinct from current approaches of modeling psychiatric illnesses in animals in two important ways. First, it proposes to replace the diagnostic and statistical manual of mental disorders (DSM) diagnostic system with measurable endophenotypes as the basis for modeling human psychopathology in animals. We argue that a major difficulty in establishing valid animal models lies in their reliance on the DSM/International Classification of Diseases conceptual framework, and suggest that the Research Domain Criteria project, recently proposed by the NIMH, provides a more suitable system to model human psychopathology in animals. Second, this proposal emphasizes the developmental origin of many (though clearly not all) psychiatric illnesses, an issue that is often glossed over in current animal models of mental illness. We suggest that animal models are essential to elucidate the mechanisms by which neurodevelopmental changes program complex behavior in adulthood. A better understanding of this issue, in animals, is the key for defining human psychopathology, and the development of earlier and more effective interventions for mental illness.

The role of early life stress in development of the anterior limb of the internal capsule in nonhuman primates

Deep brain stimulation (DBS) of the anterior limb of the internal capsule (ALIC) may be effective in treating depression. Parental verbal abuse has been linked to decreased fractional anisotropy (FA) of white matter and reduced FA correlated with depression and anxiety scores. Utilizing a nonhuman primate model of mood and anxiety disorders following disrupted mother-infant attachment, we examined whether adverse rearing conditions lead to white matter impairment of the ALIC. We examined white matter integrity using Diffusion Tensor Imaging (DTI) on a 3T-MRI. Twenty-one adult male Bonnet macaques participated in this study: 12 were reared under adverse [variable foraging demand (VFD)] conditions whereas 9 were reared under normative conditions. We examined ALIC, posterior limb of the internal capsule (PLIC) and occipital white matter. VFD rearing was associated with significant reductions in FA in the ALIC with no changes evident in the PLIC or occipital cortex white matter. Adverse rearing in monkeys persistently impaired frontal white matter tract integrity, a novel substrate for understanding affective susceptibility.

Early life stress increases anxiety-like behavior in Balbc mice despite a compensatory increase in levels of postnatal maternal care

A better understanding of the molecular and cellular mechanisms by which early life stress (ELS) modifies brain development and adult behavior is necessary for diagnosing and treating psychopathology associated with exposure to ELS. For historical reasons, most of the work in rodents has been done in rats and attempts to establish robust and reproducible paradigms in the mouse have proven to be challenging. Here we show that under normal rearing conditions, increased levels of postnatal maternal care are associated with a decrease in anxiety-like behavior in BALB/cByj offspring. Brief daily pup-dam separation (BDS) during the postnatal period was associated with increased postnatal maternal care but was surprisingly associated with increased anxiety-like behavior in adult offspring, providing the first example in which offspring receiving higher levels of postnatal maternal care are more anxious in adulthood. Plasma corticosterone levels were elevated in BDS pups even 3 h after the pups were reunited with the dam, suggesting that this paradigm represents a form of early life stress. We also show that levels of total RNA and DNA in the hippocampus reach a peak at postnatal day 14 and that exposure to BDS seems to inhibit this developmental growth spurt. We propose that exposure to stress during the postnatal period overrides the ability of high levels of postnatal maternal care to program anxiety-like behavior by inhibiting the normal growth spurt that characterizes this period.

Early life stress perturbs the maturation of microglia in the developing hippocampus

Children exposed to abuse or neglect show abnormal hippocampal development and similar findings have been reported in rodent models. Using brief daily separation (BDS), a mouse model of early life stress, we previously showed that exposure to BDS impairs hippocampal function in adulthood and perturbs synaptic maturation, synaptic pruning, axonal growth and myelination in the developing hippocampus. Given that microglia are involved in these developmental processes, we tested whether BDS impairs microglial activity in the hippocampus of 14 (during BDS) and 28-day old mice (one week after BDS). We found that BDS increased the density and altered the morphology of microglia in the hippocampus of 14-day old pups, effects that were no longer present on postnatal day (PND) 28. Despite the normal cell number and morphology seen at PND28, the molecular signature of hippocampal microglia, assessed using the NanoString immune panel, was altered at both ages. We showed that during normal hippocampal development, microglia undergo significant changes between PND14 and PND28, including reduced cell density, decreased ex vivo phagocytic activity, and an increase in the expression of genes involved in inflammation and cell migration. However, microglia harvested from the hippocampus of 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 for most of the BDS-induced changes at PND14 and PND28. These findings are the first to demonstrate that early life stress dysregulates microglial function in the developing hippocampus and to identify key transcription factors that are likely to mediate these changes.

Arguable Assumptions, Debatable Conclusions

To the Editor: Munafo et al. (1) conducted a meta-analysis of a subset of studies that examined the interaction between the serotonin transporter (5-HTTLPR) gene and stressful life events (SLEs) in conferring risk for depression. 5-HTTLPR has two common alleles of varying length, which are designated “s” for short and “l” for long. They concluded that the prior “positive results for the 5-HTTLPR × SLE interactions in logistic regression models are compatible with chance findings” and stated further that “the 5-HTTLPR and SLE interaction effect is negligible.” These conclusions, however, were based on assumptions in the simulation model that are inconsistent with prior research findings and a bias in sampling strategy that call into question the authors’ conclusions. In the simulation reported in the Munafo article, the environmental effect was specified as having two levels—unexposed (E1) and exposed (E2)—corresponding to zero and one + SLE. As can be seen in Figure 1, in the original report by Caspi et al. (2) the presence of only one SLE conferred no risk for depression, regardless of genotype. Risk for depression in association with the “s” allele of 5-HTTLPR only began to increase significantly in the presence of three or more SLEs and was most pronounced in individuals with four or more SLEs. Collapsing subjects across the various levels of exposure to SLEs added significant noise in the model and compromised efforts to detect the 5-HTTLPR × SLE interaction in the meta-analysis. Figure 1 Results of multiple regression analyses estimating the association between number of stressful life events (between ages 21 years and 26 years) and the probability of major depressive disorder by age 26 as a function of serotonin transporter protein genotype. ... The nonrepresentative nature of the sample of studies included in the meta-analysis is also a limitation of this report. Munafo et al. identified 14 studies of 33 published reports that met their criteria for inclusion in the meta-analysis but were unfortunately only able to obtain data from 5 of the 14 studies. The meta-analysis was conducted with data from less than one-half the studies that met the stated inclusion criteria, with a trend for negative studies to be over-represented in the meta-analysis. Of the 14 studies meeting the inclusion criteria for the meta-analysis, 75% (3/4) of the negative studies and only 20% (2/10) of the positive studies were included in the analysis (Fisher exact test: p < .10). There has been a proliferation of studies examining this gene × environment interaction over the past 2 years and, to date, no comprehensive meta-analysis of all studies. Since the publication of the article by Munafo et al., a second meta-analysis was published that similarly concluded that there is no evidence that the serotonin transporter genotype alone or in interaction with SLEs is associated with an elevated risk for depression (3). Although a larger number of studies were included in the second meta-analysis, 12 studies were published after the dataset for this second meta-analysis was closed, with only 3 of the 12 studies including adequate data in the published reports to be examined in the second meta-analysis (K.R. Merikangas, written communication, July 2009). Seven of the nine studies excluded because they were published after the dataset was closed were full or partial replications (4–10), as were four other earlier studies that were not included in the second meta-analysis because the data were either not received or received in an incompatible format (11–14). Like the publication by Munafo et al., the second meta-analysis also included an excess representation of nonreplications. It was conducted with data from one-half of the 26 studies that met the inclusion criteria for the meta-analysis (3), with a trend again for negative studies to be over-represented. Of the 26 studies meeting the inclusion criteria for the second meta-analysis, 78% (7/9) of the negative studies and only 35% (6/17) of the positive studies were included in the analysis (Fisher exact test: p < .10). Most studies in the literature (17/26) have replicated in part or fully the finding that individuals with the “s” allele of 5-HTTLPR are at higher risk for depression after exposure to multiple SLEs or experiences of child maltreatment, but to date a comprehensive meta-analysis of published results has not been completed. The serotonin transporter is a protein critical to the regulation of serotonin levels in the brain, on the basis of its ability to remove serotonin from the synapse and terminate its action. Serotonin transporter protein (5-HTT) knockout mice show stress-induced anxious- and depressive-like behaviors and increased dendritic spine density in the amygdala (15). This latter finding is interesting in light of the fact that the “s” allele of 5-HTTLPR has been associated with increased amygdala activation to negative stimuli in multiple imaging genomic studies (16). In experimental stress-induction paradigms, the “s” allele of 5-HTTLPR has also been associated with increased hypothalamic pituitary adrenal axis activity in both humans (17) and nonhuman primates (18) and heightened brain activity in stress-responsive brain regions (19). A comprehensive meta-analysis is required to determine the strength of the 5-HTTLPR × SLE interaction in conferring risk for depression. In addition, we believe more work is needed to unravel the mechanisms by which stress might confer vulnerability to depression in individuals with the “s” allele of 5-HTTLPR, to further characterize allelic variants of this gene, and identify additional genetic (e.g., BDNF) and environmental (e.g., social supports) factors that can modify the magnitude of the 5-HTTLPR × SLE interaction.

The Silent Epidemic of Neurodevelopmental Injuries

Childhood maltreatment in the United States was recently recognized as a major public health problem by several influential sources including the World Health Organization and the Institute of Medicine (1,2). An extensive survey conducted by the National Incidence of Child Abuse and Neglect (NIS3) revealed that roughly 1.5 million children were abused or neglected in 1993. This number of documented cases most likely underestimates the true prevalence, given that many cases of maltreatment go unrecognized (2). Moreover, according to the three available NIS reports, the incidence of childhood maltreatment has been steadily increasing over the past 3 decades (see also NIS4 for a good summary of this issue). Although increased reporting might explain some of the data, it is unlikely to explain this alarming trend fully (3). In the absence of effective interventions, maltreated children go on to develop a host of behavioral, emotional, cognitive, and medical sequelae that are chronic and in many cases refractory to treatment (2,4–6). The relationship between early life adversity (ELA) and mental illness has now been documented with both retrospective and prospective studies (reviewed in [2]), and several reports have consistently documented that more than one-half (!) of the individuals with chronic mental illness have been physically, verbally, or sexually abused early in life (7,8). Although most clinicians and researchers will endorse the notion that ELA is associated with increased risk for chronic mental illness, few appreciate the true magnitude of this problem. Better awareness of the burden that exposure to ELA places on adult psychiatric services represents the first step necessary towards transforming current psychiatric interventions. This paradigm shift should substitute the current focus on symptom-reduction with one that focuses on prevention and incorporates a neurodevelopmental framework to diagnose and treat ELA-associated psychopathology. These interventions will require a sound biological understanding of normal neurodevelopment and how ELA interferes with this process.

Early-Life Stress Perturbs Key Cellular Programs in the Developing Mouse Hippocampus

Conflicting reports are available with regard to the effects of childhood abuse and neglect on hippocampal function in children. While earlier imaging studies and some animal work have suggested that the effects of early-life stress (ELS) manifest only in adulthood, more recent studies have documented impaired hippocampal function in maltreated children and adolescents. Additional work using animal modes is needed to clarify the effects of ELS on hippocampal development. In this regard, genomic, proteomic, and molecular tools uniquely available in the mouse make it a particularly attractive model system to study this issue. However, very little work has been done so far to characterize the effects of ELS on hippocampal development in the mouse. To address this issue, we examined the effects of brief daily separation (BDS), a mouse model of ELS that impairs hippocampal-dependent memory in adulthood, on hippocampal development in 28-day-old juvenile mice. This age was chosen because it corresponds to the developmental period in which human imaging studies have revealed abnormal hippocampal development in maltreated children. Exposure to BDS caused a significant decrease in the total protein content of synaptosomes harvested from the hippocampus of 28-day-old male and female mice, suggesting that BDS impairs normal synaptic development in the juvenile hippocampus. Using a novel liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM) assay, we found decreased expression of many synaptic proteins, as well as proteins involved in axonal growth, myelination, and mitochondrial activity. Golgi staining in 28-day-old BDS mice showed an increase in the number of immature and abnormally shaped spines and a decrease in the number of mature spines in CA1 neurons, consistent with defects in synaptic maturation and synaptic pruning at this age. In 14-day-old pups, BDS deceased the expression of proteins involved in axonal growth and myelination, but did not affect the total protein content of synaptosomes harvested from the hippocampus, or protein levels of other synaptic markers. These results add two important findings to previous work in the field. First, our findings demonstrate that in 28-day-old juvenile mice, BDS impairs synaptic maturation and reduces the expression of proteins that are necessary for axonal growth, myelination, and mitochondrial function. Second, the results suggest a sequential model in which BDS impairs normal axonal growth and myelination before it disrupts synaptic maturation in the juvenile hippocampus.