Sex-Specific Distributed White Matter Microarchitectural Alterations in Preadolescent Youths With Anxiety Disorders: A Mega-Analytic Study.

OBJECTIVE: Anxiety disorders are among the most common psychiatric disorders in youths and emerge during childhood. This is also a period of rapid white matter (WM) development, which is critical for efficient neuronal communication. Previous work in preadolescent children with anxiety disorders demonstrated anxiety disorder-related reductions in WM microstructural integrity (fractional anisotropy [FA]) in the uncinate fasciculus (UF), the major WM tract facilitating prefrontal cortical-limbic structural connectivity. Importantly, this association was found only in boys with anxiety disorders. To confirm this finding and more comprehensively understand WM changes in childhood anxiety, this mega-analytic study characterizes WM alterations related to anxiety disorders and sex in the largest sample of preadolescent children to date. METHODS: Diffusion tensor imaging data from published studies of preadolescent children with anxiety disorders and healthy volunteers (ages 8-12) (N=198) were combined with a new data set (N=97) for a total sample of 165 children with anxiety disorders and 132 healthy volunteers. Children with anxiety disorders met DSM-5 criteria for current generalized, separation, and/or social anxiety disorder. Analyses of tractography and voxel-wise data assessed between-group differences (anxiety disorder vs. healthy volunteer), effects of sex, and their interaction. RESULTS: Tract-based and voxel-wise analyses confirmed a significant reduction in UF FA in boys but not girls with anxiety disorders. Results also demonstrated other significant widespread anxiety disorder-related WM alterations specifically in boys, including in multiple commissural, association, projection, and brainstem regions. CONCLUSIONS: In addition to confirming male-specific anxiety disorder-related reductions in UF FA, the results demonstrate that anxiety disorders in boys and not girls are associated with broadly distributed WM alterations across the brain. These findings support further studies focused on understanding the extent to which WM alterations in boys with anxiety disorders are involved in pathophysiological processes that mediate anxiety disorders. The findings also suggest the possibility that WM microarchitecture could serve as a novel treatment target for childhood anxiety disorders.

Early life adversity in primates: Behavioral, endocrine, and neural effects.

BACKGROUND: Evidence suggests that early life adversity is associated with maladaptive behaviors and is commonly an antecedent of stress-related psychopathology. This is particularly relevant to rearing in primate species as infant primates depend on prolonged, nurturant rearing by caregivers for normal development. To further understand the consequences of early life rearing adversity, and the relation among alterations in behavior, physiology and brain function, we assessed young monkeys that had experienced maternal separation followed by peer rearing with behavioral, endocrine and multimodal neuroimaging measures. METHODS: 50 young rhesus monkeys were studied, half of which were rejected by their mothers and peer reared, and the other half were reared by their mothers. Assessments were performed at approximately 1.8 years of age and included: threat related behavioral and cortisol responses, cerebrospinal fluid (CSF) measurements of oxytocin and corticotropin releasing hormone (CRH), and multimodal neuroimaging measures (anatomical scans, resting functional connectivity, diffusion tensor imaging, and threat-related regional glucose metabolism). RESULTS: The results demonstrated alterations across behavioral, endocrine, and neuroimaging measures in young monkeys that were reared without their mothers. At a behavioral level in response to a potential threat, peer reared animals engaged in significantly less freezing behavior (p = 0.022) along with increased self-directed behaviors (p < 0.012). Levels of oxytocin in the CSF, but not plasma, were significantly reduced in the peer reared animals (p = 0.019). No differences in plasma cortisol or CSF CRH were observed. Diffusion tensor imaging revealed significantly decreased white matter density across the brain. Exploratory correlational and permutation analyses suggest that the impact of peer rearing on behavior, endocrine and brain structural alterations are mediated by separate parallel mechanisms. CONCLUSIONS: Taken together, these results demonstrate in NHPs the importance of maternal rearing on the development of brain, behavior and hormonal systems that are linked to social functioning and adaptive responses. The findings suggest that the effects of maternal deprivation are mediated via multiple independent pathways which may account for the heterogeneity in behavioral and biological alterations observed in individuals that have experienced this early life adversity.

A preliminary study of the effects of an antimuscarinic agent on anxious behaviors and white matter microarchitecture in nonhuman primates.

Myelination subserves efficient neuronal communication, and alterations in white matter (WM) microstructure have been implicated in numerous psychiatric disorders, including pathological anxiety. Recent work in rodents suggests that muscarinic antagonists may enhance myelination with behavioral benefits; however, the neural and behavioral effects of muscarinic antagonists have yet to be explored in non-human primates (NHP). Here, as a potentially translatable therapeutic strategy for human pathological anxiety, we present data from a first-in-primate study exploring the effects of the muscarinic receptor antagonist solifenacin on anxious behaviors and WM microstructure. 12 preadolescent rhesus macaques (6 vehicle control, 6 experimental; 8F, 4M) were included in a pre-test/post-test between-group study design. The experimental group received solifenacin succinate for ~60 days. Subjects underwent pre- and post-assessments of: 1) anxious temperament (AT)-related behaviors in the potentially threatening no-eye-contact (NEC) paradigm (30-min); and 2) WM and regional brain metabolism imaging metrics, including diffusion tensor imaging (DTI), quantitative relaxometry (QR), and FDG-PET. In relation to anxiety-related behaviors expressed during the NEC, significant Group (vehicle control vs. solifenacin) by Session (pre vs. post) interactions were found for freezing, cooing, and locomotion. Compared to vehicle controls, solifenacin-treated subjects exhibited effects consistent with reduced anxiety, specifically decreased freezing duration, increased locomotion duration, and increased cooing frequency. Furthermore, the Group-by-Session-by-Sex interaction indicated that these effects occurred predominantly in the males. Exploratory whole-brain voxelwise analyses of post-minus-pre differences in DTI, QR, and FDG-PET metrics revealed some solifenacin-related changes in WM microstructure and brain metabolism. These findings in NHPs support the further investigation of the utility of antimuscarinic agents in targeting WM microstructure as a means to treat pathological anxiety.

DREADD-mediated amygdala activation is sufficient to induce anxiety-like responses in young nonhuman primates.

Anxiety disorders are among the most prevalent psychiatric disorders, with symptoms often beginning early in life. To model the pathophysiology of human pathological anxiety, we utilized Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) in a nonhuman primate model of anxious temperament to selectively increase neuronal activity of the amygdala. Subjects included 10 young rhesus macaques; 5 received bilateral infusions of AAV5-hSyn-HA-hM3Dq into the dorsal amygdala, and 5 served as controls. Subjects underwent behavioral testing in the human intruder paradigm following clozapine or vehicle administration, prior to and following surgery. Behavioral results indicated that clozapine treatment post-surgery increased freezing across different threat-related contexts in hM3Dq subjects. This effect was again observed approximately 1.9 years following surgery, indicating the long-term functional capacity of DREADD-induced neuronal activation. [11C]deschloroclozapine PET imaging demonstrated amygdala hM3Dq-HA specific binding, and immunohistochemistry revealed that hM3Dq-HA expression was most prominent in basolateral nuclei. Electron microscopy confirmed expression was predominantly on neuronal membranes. Together, these data demonstrate that activation of primate amygdala neurons is sufficient to induce increased anxiety-related behaviors, which could serve as a model to investigate pathological anxiety in humans.

Gene expression in the primate orbitofrontal cortex related to anxious temperament.

Anxiety disorders are among the most prevalent psychiatric disorders, causing significant suffering and disability. Relative to other psychiatric disorders, anxiety disorders tend to emerge early in life, supporting the importance of developmental mechanisms in their emergence and maintenance. Behavioral inhibition (BI) is a temperament that emerges early in life and, when stable and extreme, is linked to an increased risk for the later development of anxiety disorders and other stress-related psychopathology. Understanding the neural systems and molecular mechanisms underlying this dispositional risk could provide insight into treatment targets for anxiety disorders. Nonhuman primates (NHPs) have an anxiety-related temperament, called anxious temperament (AT), that is remarkably similar to BI in humans, facilitating the design of highly translational models for studying the early risk for stress-related psychopathology. Because of the recent evolutionary divergence between humans and NHPs, many of the anxiety-related brain regions that contribute to psychopathology are highly similar in terms of their structure and function, particularly with respect to the prefrontal cortex. The orbitofrontal cortex plays a critical role in the flexible encoding and regulation of threat responses, in part through connections with subcortical structures like the amygdala. Here, we explore individual differences in the transcriptional profile of cells within the region, using laser capture microdissection and single nuclear sequencing, providing insight into the molecules underlying individual differences in AT-related function of the pOFC, with a particular focus on previously implicated cellular systems, including neurotrophins and glucocorticoid signaling.

Prefrontal influences on the function of the neural circuitry underlying anxious temperament in primates.

Anxious temperament, characterized by heightened behavioral and physiological reactivity to potential threat, is an early childhood risk factor for the later development of stress-related psychopathology. Using a well-validated nonhuman primate model, we tested the hypothesis that the prefrontal cortex (PFC) is critical in regulating the expression of primate anxiety-like behavior, as well as the function of subcortical components of the anxiety-related neural circuit. We performed aspiration lesions of a narrow 'strip' of the posterior orbitofrontal cortex (OFC) intended to disrupt both cortex and axons entering, exiting and coursing through the pOFC, particularly those of the uncinate fasciculus (UF), a white matter tract that courses adjacent to and through this region. The OFC is of particular interest as a potential regulatory region because of its extensive reciprocal connections with amygdala, other subcortical structures and other frontal lobe regions. We validated this lesion method by demonstrating marked lesion-induced decreases in the microstructural integrity of the UF, which contains most of the fibers that connect the ventral PFC with temporal lobe structures as well as with other frontal regions. While the lesions resulted in modest decreases in threat-related behavior, they substantially decreased metabolism in components of the circuit underlying threat processing. These findings provide evidence for the importance of structural connectivity between the PFC and key subcortical structures in regulating the functions of brain regions known to be involved in the adaptive and maladaptive expression of anxiety.

DREADD-mediated amygdala activation is sufficient to induce anxiety-like responses in young nonhuman primates.

Anxiety disorders are among the most prevalent psychiatric disorders, with symptoms often beginning early in life. To model the pathophysiology of human pathological anxiety, we utilized Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) in a nonhuman primate model of anxious temperament to selectively increase neuronal activity of the amygdala. Subjects included 10 young rhesus macaques; 5 received bilateral infusions of AAV5-hSyn-HA-hM3Dq into the dorsal amygdala, and 5 served as controls. Subjects underwent behavioral testing in the human intruder paradigm following clozapine or vehicle administration, prior to and following surgery. Behavioral results indicated that clozapine treatment post-surgery increased freezing across different threat-related contexts in hM3Dq subjects. This effect was again observed approximately 1.9 years following surgery, indicating the long-term functional capacity of DREADD-induced neuronal activation. [11C]deschloroclozapine PET imaging demonstrated amygdala hM3Dq-HA specific binding, and immunohistochemistry revealed that hM3Dq-HA expression was most prominent in basolateral nuclei. Electron microscopy confirmed expression was predominantly on neuronal membranes. Together, these data demonstrate that activation of primate amygdala neurons is sufficient to induce increased anxiety-related behaviors, which could serve as a model to investigate pathological anxiety in humans.

Evidence in primates supporting the use of chemogenetics for the treatment of human refractory neuropsychiatric disorders.

Non-human primate (NHP) models are essential for developing and translating new treatments that target neural circuit dysfunction underlying human psychopathology. As a proof-of-concept for treating neuropsychiatric disorders, we used a NHP model of pathological anxiety to investigate the feasibility of decreasing anxiety by chemogenetically (DREADDs [designer receptors exclusively activated by designer drugs]) reducing amygdala neuronal activity. Intraoperative MRI surgery was used to infect dorsal amygdala neurons with AAV5-hSyn-HA-hM4Di in young rhesus monkeys. In vivo microPET studies with [11C]-deschloroclozapine and postmortem autoradiography with [3H]-clozapine demonstrated selective hM4Di binding in the amygdala, and neuronal expression of hM4Di was confirmed with immunohistochemistry. Additionally, because of its high affinity for DREADDs, and its approved use in humans, we developed an individualized, low-dose clozapine administration strategy to induce DREADD-mediated amygdala inhibition. Compared to controls, clozapine selectively decreased anxiety-related freezing behavior in the human intruder paradigm in hM4Di-expressing monkeys, while coo vocalizations and locomotion were unaffected. These results are an important step in establishing chemogenetic strategies for patients with refractory neuropsychiatric disorders in which amygdala alterations are central to disease pathophysiology.

Transcriptional Profiling of Primate Central Nucleus of the Amygdala Neurons to Understand the Molecular Underpinnings of Early-Life Anxious Temperament.

BACKGROUND: Children exhibiting extreme anxious temperament (AT) are at an increased risk for developing anxiety and depression. Our previous mechanistic and neuroimaging work in young rhesus monkeys linked the central nucleus of the amygdala to AT and its underlying neural circuit. METHODS: Here, we used laser capture microscopy and RNA sequencing in 47 young rhesus monkeys to investigate AT's molecular underpinnings by focusing on neurons from the lateral division of the central nucleus of the amygdala (CeL). RNA sequencing identified numerous AT-related CeL transcripts, and we used immunofluorescence (n = 3) and tract-tracing (n = 2) methods in a different sample of monkeys to examine the expression, distribution, and projection pattern of neurons expressing one of these transcripts. RESULTS: We found 555 AT-related transcripts, 14 of which were confirmed with high statistical confidence (false discovery rate < .10), including protein kinase C delta (PKCδ), a CeL microcircuit cell marker implicated in rodent threat processing. We characterized PKCδ neurons in the rhesus CeL, compared its distribution with that of the mouse, and demonstrated that a subset of these neurons project to the laterodorsal bed nucleus of the stria terminalis. CONCLUSIONS: These findings demonstrate that CeL PKCδ is associated with primate anxiety, provides evidence of a CeL to laterodorsal bed nucleus of the stria terminalis circuit that may be relevant to understanding human anxiety, and points to specific molecules within this circuit that could serve as potential treatment targets for anxiety disorders.

Dorsal Amygdala Neurotrophin-3 Decreases Anxious Temperament in Primates.

BACKGROUND: An early-life anxious temperament (AT) is a risk factor for the development of anxiety, depression, and comorbid substance abuse. We validated a nonhuman primate model of early-life AT and identified the dorsal amygdala as a core component of AT's neural circuit. Here, we combine RNA sequencing, viral-vector gene manipulation, functional brain imaging, and behavioral phenotyping to uncover AT's molecular substrates. METHODS: In response to potential threat, AT and brain metabolism were assessed in 46 young rhesus monkeys. We identified AT-related transcripts using RNA-sequencing data from dorsal amygdala tissue (including central nucleus of the amygdala [Ce] and dorsal regions of the basal nucleus). Based on the results, we overexpressed the neurotrophin-3 gene, NTF3, in the dorsal amygdala using intraoperative magnetic resonance imaging-guided surgery (n = 5 per group). RESULTS: This discovery-based approach identified AT-related alterations in the expression of well-established and novel genes, including an inverse association between NTRK3 expression and AT. NTRK3 is an interesting target because it is a relatively unexplored neurotrophic factor that modulates intracellular neuroplasticity pathways. Overexpression of the transcript for NTRK3's endogenous ligand, NTF3, in the dorsal amygdala resulted in reduced AT and altered function in AT's neural circuit. CONCLUSIONS: Together, these data implicate neurotrophin-3/NTRK3 signaling in the dorsal amygdala in mediating primate anxiety. More generally, this approach provides an important step toward understanding the molecular underpinnings of early-life AT and will be useful in guiding the development of treatments to prevent the development of stress-related psychopathology.

Ultrastructural localization of DREADDs in monkeys.

Designer receptors exclusively activated by designer drugs (DREADDs) are extensively used to modulate neuronal activity in rodents, but their use in primates remains limited. An essential need that remains is the demonstration that DREADDs are efficiently expressed on the plasma membrane of primate neurons. To address this issue, electron microscopy immunogold was used to determine the subcellular localization of the AAV vector-induced DREADDs hM4Di and hM3Dq fused to different tags in various brain areas of rhesus monkeys and mice. When hM4Di was fused to mCherry, the immunogold labelling was mostly confined to the intracellular space, and poorly expressed at the plasma membrane in monkey dendrites. In contrast, the hM4Di-mCherry labelling was mostly localized to the dendritic plasma membrane in mouse neurons, suggesting species differences in the plasma membrane expression of these exogenous proteins. The lack of hM4Di plasma membrane expression may limit the functional effects of systemic administration of DREADD-actuators in monkey neurons. Removing the mCherry and fusing of hM4Di with the haemagglutinin (HA) tag resulted in strong neuronal plasma membrane immunogold labelling in both monkeys and mice neurons. Finally, hM3Dq-mCherry was expressed mostly at the plasma membrane in monkey neurons, indicating that the fusion of mCherry with hM3Dq does not hamper membrane incorporation of this specific DREADD. Our results suggest that the pattern of ultrastructural expression of DREADDs in monkey neurons depends on the DREADD/tag combination. Therefore, a preliminary characterization of plasma membrane expression of specific DREADD/tag combinations is recommended when using chemogenetic approaches in primates.

Somatostatin Gene and Protein Expression in the Non-human Primate Central Extended Amygdala.

Alterations in central extended amygdala (EAc) function have been linked to anxiety, depression, and anxious temperament (AT), the early-life risk to develop these disorders. The EAc is composed of the central nucleus of the amygdala (Ce), the bed nucleus of the stria terminalis (BST), and the sublenticular extended amygdala (SLEA). Using a non-human primate model of AT and multimodal neuroimaging, the Ce and the BST were identified as key AT-related regions. Both areas are primarily comprised of GABAergic neurons and the lateral Ce (CeL) and lateral BST (BSTL) have among the highest expression of neuropeptides in the brain. Somatostatin (SST) is of particular interest because mouse studies demonstrate that SST neurons, along with corticotropin-releasing factor (CRF) neurons, contribute to a threat-relevant EAc microcircuit. Although the distribution of CeL and BSTL SST neurons has been explored in rodents, this system is not well described in non-human primates. In situ hybridization demonstrated an anterior-posterior gradient of SST mRNA in the CeL but not the BSTL of non-human primates. Triple-labeling immunofluorescence staining revealed that SST protein-expressing cell bodies are a small proportion of the total CeL and BSTL neurons and have considerable co-labeling with CRF. The SLEA exhibited strong SST mRNA and protein expression, suggesting a role for SST in mediating information transfer between the CeL and BSTL. These data provide the foundation for mechanistic non-human primate studies focused on understanding EAc function in neuropsychiatric disorders.

Altered Uncinate Fasciculus Microstructure in Childhood Anxiety Disorders in Boys But Not Girls.

OBJECTIVE: Anxiety disorders are common, can result in lifelong suffering, and frequently begin before adolescence. Evidence from adults suggests that altered prefrontal-limbic connectivity is a pathophysiological feature of anxiety disorders. More specifically, in adults with anxiety disorders, decreased fractional anisotropy (FA), a measure of white matter integrity, has been observed in the uncinate fasciculus, the major tract that connects limbic and prefrontal regions. Because of the early onset of anxiety disorders and the increased incidence in anxiety disorders in females during their reproductive years, it is important to understand whether the reduction in uncinate fasciculus FA exists in children with anxiety disorders and the extent to which this alteration is sex related. To address these issues, the authors assessed FA in the uncinate fasciculus in unmedicated boys and girls with anxiety disorders. METHODS: FA measures were derived from diffusion tensor images that were acquired from 98 unmedicated children (ages 8-12); 52 met criteria for generalized anxiety disorder, separation anxiety disorder, social anxiety disorder, or anxiety disorder not otherwise specified, and 46 were matched control subjects. RESULTS: Tract-based results demonstrated that children with anxiety disorders have significant reductions in uncinate fasciculus FA. A significant sex-by-group interaction and post hoc testing revealed that this effect was evident only in boys. No other main effects or sex-by-group interactions were found for other white matter tracts. CONCLUSIONS: These findings provide evidence of uncinate fasciculus white matter alterations in boys with anxiety disorders. The data demonstrate that anxiety disorder-related alterations in prefrontal-limbic structural connectivity are present early in life, are not related to psychotropic medication exposure, and are sex specific. Building on these findings, future research has the potential to provide insights into the genesis and sexual dimorphism of the pathophysiology that leads to anxiety disorders, as well as to identify sex-specific early-life treatment targets.

Myelin modifications after chronic sleep loss in adolescent mice.

Study Objectives: Previous studies found that sleep loss can suppress the expression of genes implicated in myelination and can have adverse effects on oligodendrocyte precursor cells. On the other hand, sleep may favor myelination by promoting the expression of genes involved in its formation and maintenance. Albeit limited, these results suggest that sleep loss can have detrimental effects on the formation and maintenance of myelin. Methods: Here, we tested this hypothesis by evaluating ultrastructural modifications of myelin in two brain regions (corpus callosum and lateral olfactory tract) of mice exposed to different periods of sleep loss, from a few hours of sleep deprivation to ~5 days of chronic sleep restriction. In addition, we measured the internodal length-the distance between consecutive nodes of Ranvier along the axon-and plasma corticosterone levels. Results: We find that g-ratio-the ratio of the diameter of the axon itself to the outer diameter of the myelinated fiber-increases after chronic sleep loss. This effect is mediated by a reduction in myelin thickness and is not associated with changes in the internodal length. Relative to sleep, plasma corticosterone levels increase after acute sleep deprivation, but show only a trend to increase after chronic sleep loss. Conclusions: Chronic sleep loss may negatively affect myelin.

Overexpressing Corticotropin-Releasing Factor in the Primate Amygdala Increases Anxious Temperament and Alters Its Neural Circuit.

BACKGROUND: Nonhuman primate models are critical for understanding mechanisms underlying human psychopathology. We established a nonhuman primate model of anxious temperament (AT) for studying the early-life risk to develop anxiety and depression. Studies have identified the central nucleus of the amygdala (Ce) as an essential component of AT's neural substrates. Corticotropin-releasing factor (CRF) is expressed in the Ce, has a role in stress, and is linked to psychopathology. Here, in young rhesus monkeys, we combined viral vector technology with assessments of anxiety and multimodal neuroimaging to understand the consequences of chronically increased CRF in the Ce region. METHODS: Using real-time intraoperative magnetic resonance imaging-guided convection-enhanced delivery, five monkeys received bilateral dorsal amygdala Ce-region infusions of adeno-associated virus serotype 2 containing the CRF construct. Their cagemates served as unoperated control subjects. AT, regional brain metabolism, resting functional magnetic resonance imaging, and diffusion tensor imaging were assessed before and 2 months after viral infusions. RESULTS: Dorsal amygdala CRF overexpression significantly increased AT and metabolism within the dorsal amygdala. Additionally, we observed changes in metabolism in other AT-related regions, as well as in measures of functional and structural connectivity. CONCLUSIONS: This study provides a translational roadmap that is important for understanding human psychopathology by combining molecular manipulations used in rodents with behavioral phenotyping and multimodal neuroimaging measures used in humans. The results indicate that chronic CRF overexpression in primates not only increases AT but also affects metabolism and connectivity within components of AT's neural circuitry.

Differentially methylated plasticity genes in the amygdala of young primates are linked to anxious temperament, an at risk phenotype for anxiety and depressive disorders.

Children with an anxious temperament (AT) are at a substantially increased risk to develop anxiety and depression. The young rhesus monkey is ideal for studying the origin of human AT because it shares with humans the genetic, neural, and phenotypic underpinnings of complex social and emotional functioning. Heritability, functional imaging, and gene expression studies of AT in young monkeys revealed that the central nucleus of the amygdala (Ce) is a key environmentally sensitive substrate of this at risk phenotype. Because epigenetic marks (e.g., DNA methylation) can be modulated by environmental stimuli, these data led us to hypothesize a role for DNA methylation in the development of AT. To test this hypothesis, we used reduced representation bisulfite sequencing to examine the cross-sectional genome-wide methylation levels in the Ce of 23 age-matched monkeys (1.3 ± 0.2 years) phenotyped for AT. Because AT reflects a continuous trait-like variable, we used an analytical approach that is consistent with this biology to identify genes in the Ce with methylation patterns that predict AT. Expression data from the Ce of these same monkeys were then used to find differentially methylated candidates linked to altered gene regulation. Two genes particularly relevant to the AT phenotype were BCL11A and JAG1. These transcripts have well-defined roles in neurodevelopmental processes, including neurite arborization and the regulation of neurogenesis. Together, these findings represent a critical step toward understanding the effects of early environment on the neuromolecular mechanisms that underlie the risk to develop anxiety and depressive disorders.

Array-based assay detects genome-wide 5-mC and 5-hmC in the brains of humans, non-human primates, and mice.

BACKGROUND: Methylation on the fifth position of cytosine (5-mC) is an essential epigenetic mark that is linked to both normal neurodevelopment and neurological diseases. The recent identification of another modified form of cytosine, 5-hydroxymethylcytosine (5-hmC), in both stem cells and post-mitotic neurons, raises new questions as to the role of this base in mediating epigenetic effects. Genomic studies of these marks using model systems are limited, particularly with array-based tools, because the standard method of detecting DNA methylation cannot distinguish between 5-mC and 5-hmC and most methods have been developed to only survey the human genome. RESULTS: We show that non-human data generated using the optimization of a widely used human DNA methylation array, designed only to detect 5-mC, reproducibly distinguishes tissue types within and between chimpanzee, rhesus, and mouse, with correlations near the human DNA level (R(2) > 0.99). Genome-wide methylation analysis, using this approach, reveals 6,102 differentially methylated loci between rhesus placental and fetal tissues with pathways analysis significantly overrepresented for developmental processes. Restricting the analysis to oncogenes and tumor suppressor genes finds 76 differentially methylated loci, suggesting that rhesus placental tissue carries a cancer epigenetic signature. Similarly, adapting the assay to detect 5-hmC finds highly reproducible 5-hmC levels within human, rhesus, and mouse brain tissue that is species-specific with a hierarchical abundance among the three species (human > rhesus >> mouse). Annotation of 5-hmC with respect to gene structure reveals a significant prevalence in the 3'UTR and an association with chromatin-related ontological terms, suggesting an epigenetic feedback loop mechanism for 5-hmC. CONCLUSIONS: Together, these data show that this array-based methylation assay is generalizable to all mammals for the detection of both 5-mC and 5-hmC, greatly improving the utility of mammalian model systems to study the role of epigenetics in human health, disease, and evolution.

Neuropeptide Y receptor gene expression in the primate amygdala predicts anxious temperament and brain metabolism.

BACKGROUND: Anxious temperament (AT) is identifiable early in life and predicts the later development of anxiety disorders and depression. Neuropeptide Y (NPY) is a putative endogenous anxiolytic neurotransmitter that adaptively regulates responses to stress and might confer resilience to stress-related psychopathology. With a well-validated nonhuman primate model of AT, we examined expression of the NPY system in the central nucleus (Ce) of the amygdala, a critical neural substrate for extreme anxiety. METHODS: In 24 young rhesus monkeys, we measured Ce messenger RNA (mRNA) levels of all members of the NPY system that are detectable in the Ce with quantitative real time polymerase chain reaction. We then examined the relationship between these mRNA levels and both AT expression and brain metabolism. RESULTS: Lower mRNA levels of neuropeptide Y receptor 1 (NPY1R) and NPY5R but not NPY or NPY2R in the Ce predicted elevated AT; mRNA levels for NPY1R and NPY5R in the motor cortex were not related to AT. In situ hybridization analysis provided for the first time a detailed description of NPY1R and NPY5R mRNA distribution in the rhesus amygdala and associated regions. Lastly, mRNA levels for these two receptors in the Ce predicted metabolic activity in several regions that have the capacity to regulate the Ce. CONCLUSIONS: Decreased NPY signaling in the Ce might contribute to the altered metabolic activity that is a component of the neural substrate underlying AT. This suggests that enhancement of NPY signaling might reduce the risk to develop psychopathology.

Central amygdala nucleus (Ce) gene expression linked to increased trait-like Ce metabolism and anxious temperament in young primates.

Children with anxious temperament (AT) are particularly sensitive to new social experiences and have increased risk for developing anxiety and depression. The young rhesus monkey is optimal for studying the origin of human AT because it shares with humans the genetic, neural, and phenotypic underpinnings of complex social and emotional functioning. In vivo imaging in young monkeys demonstrated that central nucleus of the amygdala (Ce) metabolism is relatively stable across development and predicts AT. Transcriptome-wide gene expression, which reflects combined genetic and environmental influences, was assessed within the Ce. Results support a maladaptive neurodevelopmental hypothesis linking decreased amygdala neuroplasticity to early-life dispositional anxiety. For example, high AT individuals had decreased mRNA expression of neurotrophic tyrosine kinase, receptor, type 3 (NTRK3). Moreover, variation in Ce NTRK3 expression was inversely correlated with Ce metabolism and other AT-substrates. These data suggest that altered amygdala neuroplasticity may play a role the early dispositional risk to develop anxiety and depression.