Mar M. Sanchez, Ph.D. - US grants

Several recent studies have demonstrated that central oxytocin and vasopressin pathways are important for species typical social behaviors in rodents. This proposal extends this work to non-human primates by investigating oxytocin and vasopressin in both normal and socially abnormal rhesus monkeys. Proposed studies will characterize the social and cognitive deficits in these monkeys, investigate the influence of centrally administered agonists and antagonists, and in post-mortem studies, assess oxytocin and vasopressin function with cellular and molecular techniques. As many of the behavioral deficits in these socially abnormal monkeys resemble the core features of autism and the deficit syndrome of schizophrenia, and reactive attachment disorder, the results from the proposed studies may provide important insights about the pathophysiology and novel medications for these treatment-refractory syndromes.

The Primate Animal Model Core (Core 9002) of the Emory University Center for Neuroscience of Mental Disorders (CNMD) will serve as the source of all non-human primates used by the Research Projects. The primary goal of this Core is to produce, maintain and characterize a non-human primate epigenetic early life stress (ELS) model involving neonatal exposure of rhesus monkey (M. mulatta) mother-infant dyads to social instability in the form of intermittent maternal separations. In this protocol, beginning at 3 months postpartum mothers are removed from social groups composed of 4-5 females and a single male for short (0.5, 3 or 6 hr) intervals. Both the timing and the duration of each separation are determined by a pseudo-randomized schedule. Animals experience a total of 36 separations over a 90 day period (from 3-6 months of age). Over the past 4 years, this Core has produced and characterized approximately 70 animals (approx. 20 animals per year beginning in year 02). Extensive neuroendocrine and behavioral characterization along with structural MRI is performed to determine and verify the phenotype of these animals. One important goal of this Core is to characterize developmental changes in these animals as they pass through significant transitions such as puberty. Animals are assigned to individual Conte Projects as required for specific studies of hypothalamic and extra-hypothalamic CRF systems (Project 0001), immune system function (Project 0009), acoustic startle (Project 0006), cognition and drug abuse liability (Project 0010), multimodal imaging of structural and functional neurocircuits (Project 0013). This procedure offers important advantages and sources of validity: (1) the contingencies imposed by the paradigm are naturalistic, infant development is influenced by unpredictable changes in mother's availability, (2) the demands are imposed during a critical period of development when infants solicit reassurance as they begin to explore their environment, (3) as found in some human psychopathologies, deficits are reflected in the capacity of monkeys to respond to stressful experiences as adults. Comparison of this primate model with the genetic and ELS rodent models (see Core 9001) will permit individual Research Projects to test the thesis that, in diverse species, similar neurocircuits and intracellular mechanisms are impacted by genetic and epigenetic factors across which give rise to a depressive-like syndrome. In addition to production, characterization and maintenance of these animals, Core 9003 will assist in sampling or surgical procedures, drug treatments, or necropsy as required by individual Research Projects. This organization allows consistency in animal handling and implementation of all protocols so that each Conte Research Project receives identically characterized and treated animals for study.

mental disorders; neuropsychology; Primates; animal colony;

psychopharmacology; social behavior; vasopressins; Primates; animal colony; oxytocin; behavioral /social science research tag;

corticotropin releasing factor

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have used a unique, naturalistic, nonhuman primate model of human childhood maltreatment to characterize the long-term impact of adverse caregiving on neurodevelopment using in vivo neuroimaging (structural MRI, diffusion tensor imaging (DTI) and MR spectroscopy). In order to understand the effects, we also studied normative brain development during the rhesus monkey infant period, and analyzed potential associations with socioemotional maturation. We scanned rhesus infants at 2 weeks, and 3, 6 and 12 months of age. Our preliminary data analysis revealed dramatic brain developmental changes during infancy (particularly striking between 2 weeks and 3 months), including increased brain volume, deepening of sulci and intense rates of myelination. We also found that at 2 weeks some brain cortical regions, such as the prefrontal cortex, were not yet myelinated. These findings underscored the high vulnerability of the brain to insults/adversity experienced at these early ages, which could result in alterations in normal brain developmental trajectories. This is, indeed, what we found in our studies: pathological brain development caused by adverse early caregiving. Our analyses revealed long-term alterations in brain white matter integrity in animals that received poor maternal care, specifically, increased fractional anisotropy (FA, a measure of microstructural rganization/integrity) in cortical regions such as frontal lobe, somatosensory, parietal and left visual cortices, during adolescence. In contrast, FA was reduced in the ventral striatum and right visual cortex. These results suggest that poor caregiving causes alterations in normal maturation of brain myelinated tracts. In the areas showing increased FA (e.g. prefrontal and parietal cortices), this was positively correlated with aggression and negatively with levels of serotonin. The increased FA could reflect increased myelination, which would increase the action potential speed of axons in the affected bundle, perhaps causing a mismatch in normal temporal encoding;and consequently altered behavior and physiology.

Project 3 uses a nonhuman primate model of early life stress (ELS) in the form of adverse early care to provide information on its neurodevelopmental impact using methods that cannot be used with the very young children studied in the Center's human projects (1 & 2). By nesting our rhesus project in the context of comparable work on young children exposed to ELS, and by using comparable measures of behavior and physiology across species, we are in a unique position to use the neurodevelopmental findings to understand the neural systems impacted by ELS in young primates. We have chosen a nonhuman model of adverse early care, maternal maltreatment, with striking resemblance to human maltreatment. The targeted developmental period in Project 3 is birth through 18 mos, which corresponds to the birth to 6-7 yr period targeted in our human projects. In both species this developmental period is one of rapid brain development believed to be vulnerable to ELS impact. Project 3 will assess all Center aims: (1) to test the hypothesis that ELS sensitizes developing stress- and threat-response systems and alters the development of prefrontal circuits involved in emotion- and attention-regulation; and (2) to examine the dimensions of caregiving associatedwith this sensitization of stress- and threat-response systems. Project 3 shares aims and methods with Projects 1 (foster care) and 2 (post-institutionalization). In Aim 1 stress system activity is assessed using plasma cortisol and ACTH and CSF measures of neuropeptides (CRF, AVP) and moncaminergic function; alterations in emotional behavior (fear, anxiety) are assessed from observations in the social groups and laboratory challenges; prefrontal attention- and behavior-regulatory function is assessed using tasks of attention regulation (ID/ED shift), impulsivity (Object Retrieval/Detour) and inhibitory control of behavior (Reinforcer Devaluation); effects of ELS on neurodevelopment are assessed by structural MRI, DTI and MRS. In Aim 2, caregiving behavior is assessedfocusing on the sensitivity and rejection dimensions from observations in the social groups and from videotapes using qualitative ratings. This project draws all five cores of the Center.

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (01) Behavior, Behavioral Change, and Prevention and specific Challenge Topic 03-MH-101* Biomarkers in mental disorders. The major hypothesis is that post-traumatic stress disorder (PTSD) leads to a reduction in the ability to respond to safety signals. For example, central to the clinical problem of PTSD, which has a ~90% comorbidity with major depressive disorder in patients who had early life stress, is the inability of these patients to inhibit their fear to stimuli reminiscent of their traumatic experience, even in safe conditions. In fact, we now have evidence that the inability to inhibit fear may be a unique biomarker of PTSD. Thus the sound of a car back firing or the smell of sulfur can lead to an intense fear reaction in Viet Nam veterans many years after combat, even in the safety of their own home. Despite substantial anecdotal evidence in support of this hypothesis, direct tests in humans or animals, using well-controlled behavioral paradigms, have been limited. We have now developed in rats, rhesus monkeys and humans an objective measure of safety signal learning and expression using identical paradigms and the acoustic startle reflex in all species. Three cues are used in the form AX+/BX-, where cues A and X in compound (e.g. a light and air blowing from a quiet fan) are paired with an aversive event (+), and a new cue B (e.g. a tone) and the same cue X signal no aversive event (-). Cue A becomes excitatory as the subject learns that A and X presented together predict the US. Cue B becomes inhibitory because B presented with X predicts "safety" from the US. In a critical subsequent transfer test trial, presentation of A and B together (AB) results in a reduced fear response compared with the response to A. In three independent studies PTSD patients sometimes could discriminate AX from BX while others could not. However, in all three studies PTSD patients did not inhibit fear to A on AB test trials. Because PTSD has high comorbidity with early life stress and major depression other studies in our group have now shown that patients with early life stress only or major depression only have normal safety signal learning and only patients with PTSD or PTSD with depression fail in show safety signal learning or expression. Thus, we believe we have an objective way to measure a major biomarker of PTSD. In a prior grant we tested 6 young adult rhesus monkeys, 3 with typical social and mother-infant interactions (controls) and 3 separated from their mothers for variable periods of time each day when they were infants (maternally-separated). 3 control and 1 maternally-separated showed successful AX+, BX- discrimination and all of them had less startle in the presence of AB, vs. A. The other two monkeys, both maternally separated, were never able to discriminate between A+ and B- because they continued to be fearful of both A and B (i.e. no safety signal learning following early life stress). The one maternally separated monkey that learned very well suggests resilience to early life stress in this animal. Currently we are testing the second cohort of 6 monkeys which should be completed in the next 2 months so we will have 12 monkeys in total, 6 control and 6 maternally-separated. As part of a long standing ongoing program at the Yerkes National Primate Center studying the effects of early life stress in rhesus monkeys, a great deal of data on another 36 animals in addition to the 12 we will have tested has been carried out. This includes neuroendrocrine, behavioral, autonomic, neuroimaging and genetic data, as well as banked DNA from all the animals available for further genotyping. We believe this is a tremendous resource that should not be wasted. If we cannot pay the per diems on these animals they will have to be released and we will no longer have access to them. Hence, we propose to finish testing these 36 additional monkeys so that our final sample will be 48 monkeys. This will allow a thorough analysis of how early life stress affects safety signal learning, autonomic, neuroimaging and neuroendrocrine functioning and whether some of these effects can be associated with various genetic variants. It may also detect monkeys that are resilient to early life stress so the measures already collected on them can be correlated with this behavioral measure of resilience. This is a "shovel ready" project. By hiring two new, full time technicians devoted entirely to this project we can get this work done in 2 years and believe it will be money well spent by NIMH. PUBLIC HEALTH RELEVANCE: The major hypothesis is that post-traumatic stress disorder (PTSD) leads to a reduction in the ability to respond to safety signals. We have evidence for this in humans using an objective test of fear inhibition. In this grant we want to evaluate whether early life stress in rhesus monkeys will associated with this deficit using the same objective measure of fear inhibition we also have developed in monkeys. If so would provide the first model of PTSD in rhesus monkeys.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We used a NHP model of early life stress (ELS) in the form of adverse early care to study its neurodevelopmental impact using methods that cannot be used with very young children. The targeted developmental period is birth through 18 mos, equivalent to early childhood in humans. This developmental period involves rapid brain development believed to be vulnerable to ELS impact. We collected all longitudinal measures proposed in the project for the rhesus infants born so far (n=24) at 2 weeks and 3, 6, 12 and 18 months of age. We examined the effects of ELS on emotional reactivity and have preliminary data suggesting that rhesus infants that received poor maternal care are more emotionally reactive and show deficits in cognitive functions controlled by prefrontal-limbic circuits. The increased emotional reactivity was detected from observations in the social groups and laboratory challenges (LabTAB and Human Intruder tests). We also studied the effects of ELS on the stress physiology via measurements of cortisol and ACTH plasma levels, as well as CSF levels of stress neuropeptides (CRF, AVP) and monoaminergic function, but assays are pending. When examining the caregiving dimensions associated with ELS-infants'sensitization of stress-response systems, maternal rejection seems the strongest predictor of poor infant developmental outcomes. The underlying neurodevelopmental alterations that mediate the effects of ELS on emotional behavior, stress systems and cognitive function was also assessed using neuroimaging methods (structural magnetic resonance imaging (MRI) and diffusion tensor imaging (DTI));data are pending analysis.

PROJECT SUMMARY (See instructions): The Neurochemistry Core is a dual function core that will support the research efforts ofthe Projects by providing services while at the same time developing new tools and datasets that will ultimately inform each ofthe Projects. The core will provide basic histology services to document probe placements for Projects 1 and 2, and will perform quantitative oxytocin receptor (OXTR) autoradiography in rats and voles for Project 1. Th13 Core will also provide Projects 2 and 3 with novel potent and selective OXTR antagonists for their studies in nonhuman primates. In addition, the Core will play a critical role in delineating the neuroanatomical localization of OXTR in the primate brain (currently not understood). This knowledge is essential for understanding the neural mechanisms by which oxytocin (OT) modulates social cognition in Projects 2 and 3. We will use a three pronged approach for mapping the OXTR in rhesus monkey brain. First, a validated RT-PCR technique will be used to map the location of OXTR in amygdala (Amy) subnuclei as well as to determine the neuronal phenotype of OXTR neurons after single cell recording. Second, we will work to improve the selectivity of receptor autoradiography procedures in primate tissue. Finally we will continue our effort to develop and validate novel PET ligands for in vivo imaging of OXTR in living brains. If successful, the PET ligands will contribute directly to the goals of Project 2 and 3 and may ultimately be used to inform the human studies in Project 4. These aims and the functions and services that they support are essential for the integration of research designs and interpretation of findings across projects and will provide for the first time an understanding of the neural distribution of OXTR in the primate brain. The novel OXTR antagonists and PET' ligands will have a tremendous benefit for the entire research community investigating the role of OT on social behavior.

DESCRIPTION (provided by applicant): Studies of both animals and children show that postnatal stress may have lasting effects on brain structure and function, resulting in behavioral and cognitive impairments, particularly for females. It is also unclear how social stress experienced by the mother during gestation synergizes with postnatal stress experienced by her offspring to produce these phenotypes. Importantly, other environmental factors that may interact with stressor exposure to affect brain development during childhood are frequently overlooked, most notably the consumption of calorically dense diets (CDDs) and the resulting metabolic phenotype. Indeed, there is likely a synergy, as chronic social stress is a cumulative risk factor for childhood obesity. Not only may obesity accelerate the tempo of puberty but limited data in children suggest the developing brain is vulnerable to these metabolic insults, as increased body fat is associated with altered brain structure and deficits in cognition and emotional processing. Understanding the impact of stress and obesity on neurodevelopment is critically relevant, given alarming rates of obesity in children, likely due to the consumption of CDDs - a dietary environment quite unlike the typical low caloric diets fed animals used as models for children. Key biological signals could be stress-induced elevations in cortisol and proinflammatory cytokines that are exacerbated by increased fat mass. Prospective studies of the developmental origins of health and disease are difficult to do in children. However, socially housed rhesus monkeys provide an effective translational model, as social subordination produces distinct stress-related phenotypes even during development. This application will address four specific aims to test the overarching hypothesis that prenatal maternal stress interacts with post natal social stress to alter female neurobehavioral development from infancy through puberty and these impairments are exacerbated by obesity. Aim 1 will determine whether increased fat mass interacts with postnatal social stress to alter developmental trajectories of female social and emotional behavior, as well as prefrontal-related cognitive function. Using neuroimaging, Aim 2 will test the hypothesis that social stress and increased fat mass will synergize to alter structural and functional development of the prefrontal cortex (PFC) and its connectivity with regions regulating social and emotional behaviors as well as executive function and self-regulation from infancy, with differences accelerating through the pubertal transition. Mediation analysis in Aim 3 will examine whether cortisol and inflammatory markers mediate the effects of social stress and fat mass on impaired neurobehavioral development. Using cross-fostering, Aim 4 will determine how maternal stress during gestation synergizes with postnatal social stress and obesity to further compromise neurobehavioral development. The project will identify potential biological signals that mediate the adverse effects of stress ad obesity on brain health and behavior and, in doing so, will provide crucial information that will help shape clinical interventions and social policy improvement to optimize neurobehavioral development in girls.

DESCRIPTION (provided by applicant): Adolescence is a period of increased vulnerability for the development of substance abuse, including cocaine addiction. Despite the known risk of adolescence initiation of cocaine abuse for lifelong addiction, and its tremendous health and societal costs in the US, the neurobiological mechanisms of increased risk during this developmental period are poorly understood. The proposed studies will examine this question in a novel and highly translational adolescent nonhuman primate model, investigating the effect of an important risk/vulnerability factor: exposure to early life stress. We will also determine whether increased emotional/stress reactivity increases vulnerability to cocaine addiction, including relapse, in females. We will use a highly translational macaque model of early life stress (infant maltreatment) to examine the neurobiological mechanisms underlying increased vulnerability to cocaine abuse and relapse during adolescence. The project will build on ongoing longitudinal studies of developmental alterations exhibited by the maltreated animals, which have been characterized by our group since birth using a unique cross fostering design that rules out confounding effects of heritability on outcome measures. We have evidence that the adverse experience leads to increased emotional reactivity and alterations of prefrontal connectivity (both structural and functional) during the infant period, and we will now examine whether these alterations (1) persist during adolescence and (2) underlie increased risk to cocaine abuse. Our goal is to investigate the neurobiological mechanisms underlying increased vulnerability to cocaine abuse during adolescence in animals with a well-documented history of early life stress, with a particular focus on alterations in the dopaminergic and serotonergic systems and prefrontal connectivity with the striatum and amygdala. We hypothesize that the increased emotional reactivity/anxiety characteristic of maltreated animals exacerbates cocaine self-administration and reinstatement, and that females will be more vulnerable than males. The study will also test a pharmacological intervention, through the use of pharmacological blockade of the 5-HT2A receptor during cocaine abstinence to reduce the risk of relapse. A critical aspect of this proposal is its focus on adolescence, as it is the developmental period when humans initiate drug consumption and has been rarely examined in nonhuman primate studies of cocaine abuse.

Abstract: The clinical spectrum of hippocampal (HIPPO) dysfunction encompasses a wide range of neurological, behavioral, cognitive symptoms in various psychopathological states, and importantly developmental neuropsychiatric disorders (Schizophrenia, Autism Spectrum Disorders, anxiety, post-traumatic disorders). Thus, the study of HIPPO and, in particular, of its interactions with dorsolateral prefrontal cortex (dlPFC) has become of major interest to further understand the neurobiology of developmental neuropsychiatric disorders in which both neural regions are affected and associated with memory impairment that are generally refractory to treatment. Although rodent and nonhuman primate models have proposed that HIPPO-dlPFC disconnection is an ideal systems-level phenotype that can be used for translation to neuropsychiatric diseases, these studies have been done in fully mature subjects limiting their translation to human disorders that emerge during development. A more meaningful approach would be to assess the critical developmental periods of HIPPO- dlPFC interactions and the consequences of their dysfunction across development. We propose to trace the development of HIPPO-dlPFC interactions in monkeys from pre-adolescence to adolescence, focusing on critical cognitive functions, i.e. episodic and working memory associated with HIPPO and dlPFC, respectively. At five age periods (pre-puberty: 18-30 mo, peri-puberty: 32-37 mo, 37-42 mo, 43-47 mo, and post-puberty: 52- 58 mo), we will measure HIPPO-dependent relational memory (object-in-place memory task) and PFC- dependent working memory (serial order memory task) in 15 male monkeys (Aim 1) in parallel to underlying developmental changes in HIPPO-dlPFC structural and functional connectivity (Aim 2), using noninvasive neuroimaging techniques (structural MRI, diffusion tensor imaging and resting state functional MRI). Aim 1 will provide the timing of strengthening of memory during peri-pubertal period and Aim 2 will indicate whether the memory changes are linked to changes in strength of PFC-HIPPO connections. In Aim 3, we will use six new pre-adolescent male monkeys for a transient HIPPO-dlPFC disconnection study. By combining HIPPO- inactivation in one hemisphere and dlPFC-inactivation in the other hemisphere, via muscimol (GABA-A agonist) injections, we will demonstrate that functional HIPPO-dlPFC interactions (Aim 2) are necessary for the emergence of adult-performance (Aim1). To control for pubertal effects on measures of the 3 aims, blood gonadal hormone and sexual morphological measures will be taken and used as predictors to assess the role of pubertal age on cognitive and neural changes. The proposed studies are novel, have high translational value, and will provide a new model system to carefully and systematically study the development of HIPPO- dlPFC interactions and the cognitive consequences of their derailment in adolescence and adulthood, avoiding confounding factors (pubertal age, cross-sectional studies etc) usually affecting data on human adolescents.

PROJECT SUMMARY (Bioanalytic Core, Sanchez) The Bioanalytic Core is a multifunctional core that will support the research efforts of each of the Projects by providing histological services, developing new innovative techniques and assays, and by training members of the Center to improve their own technical skills. The Aims of the Bioanalytic Core are: (1) To Provide basic neurohistology services for verification of probe placement or injection sites for Projects 1-4, including brain sectioning, histological staining, and immunohistochemistry for viral vector detection, and microscopy for reconstruction of recording and injection sites. (2) To provide assay services and to develop new techniques for molecular characterization of specific cell types, and for validating novel CRISPR viral vectors for Projects 1-4. These services include OXTR autoradiography, and the highly sensitive RNAScope mRNA in situ hybridization (ISH) technique to co-localize mRNA in cells for the purpose of molecular phenotyping. This technique will allow for the co-localization of mRNA for OXTR with other neuromodulator systems such as dopamine D1 and D2 receptors or with choline acetyltransferase (ChAT). We will also develop PCR/sequencing or Western assays to validate viral vector mediated CRISPR editing of OXTR or OT gene for Projects 1 and 3. Finally, the core will perform genotyping for OXTR polymorphisms in prairie voles in support of Projects 1 and 2. (3) To provide training in the techniques used in the Core to make sure that students, postdoctoral fellows, staff and trainees of the Center gain experience in basic histology and staining, autoradiography, ISH, IHC, neuroanatomy and fluorescent microscopy, which will be useful for their future career development. The services of the Bioanalytic Core will facilitate progress in every project and will serve as the center memory for techniques as trainees come and go. We will also develop new assays as needed and will perform basic neurohistology with standardization across projects while freeing up time for trainees to make progress in their own projects. The Bioanalytic Core is supported by the strong neuroanatomical expertise of the PI (Sanchez) in mapping neuropeptide and steroid receptors in the primate brain, studies performed in collaboration with the Center PI (Young), who has additional expertise with OT receptor systems both in rodent and primate brain. Co-investigator Kiyoshi Inoue brings critical extensive expertise in situ hybridization using RNAScope, OXTR autoradiography, general histology for probe anatomical verification, immunohistochemistry, and genotyping the prairie vole OXTR gene. These services will facilitate the integrated research programs of the Conte Center by providing unified and standardized services for each of the Projects, and allow investigators for the first time to identify the molecular phenotype of neurons activated by the OT system, and facilitate validation of new techniques such as optogenetic transgene expression and CRISPR editing of the OT system. These services will streamline the research activity of the Projects to maximize their productivity, while also providing valuable training on each of the techniques used to all Conte personnel who wish to learn these techniques.

Abstract: A strong link between early life stress/adversity (ELS/ELA) and age-related disorders, such as cardiometabolic disease, cognitive and psychiatric/neurological disorders, have been established mostly based on retrospective human reports. Yet, prospective, longitudinal, studies across the life span are critical to identify biomarkers of ELA risk embedded earlier in life, during middle age to develop early intervention strategies. Animal models with short life spans (invertebrates, rodents) have significant limitations to inform about human aging and the therapeutics developed from those models have failed in clinical trials. Longitudinal nonhuman primate (NHP) studies could provide significant information on early biological and neural markers of ELA-related cognitive decline due to their long life span and gradual aging-related cognitive impairments and brain pathology similar to those in humans emerging in middle age. This proposal builds on our data linking ELA in macaques with early markers of accelerated cellular aging (accelerated DNA methylation age, shortened telomere length), inflammation, and neurocognitive alterations detectable from infancy to young adulthood. The goal is to use a prospective, longitudinal design in NHPs to identify early biomarkers/pathways and underlying mechanisms of ELA-related accelerated neural aging trajectories and cognitive decline from young adulthood to middle age. This unique population of adult female macaques with ELA (social subordination stress) are currently living in social groups at the Yerkes National Primate Research Center and were longitudinally characterized from birth through puberty as part of NIH-funded studies. These animals (High-ELA: subordinates; low-ELA: dominant) will be studied longitudinally between 7(early adulthood) and 11 (middle age) years of age. Aim 1 will examine trajectories of ELA-accelerated neural aging in brain regions that control cognitive and stress/emotional functions studied in Aim 2 (prefrontal cortex -PFC-, hippocampus -HIPP-, amygdala); it will use (a) MRI, DTI and resting state fMRI to examine myelin loss, cortical thinning and loss of long-range connectivity; (b) measures of neuropathology with MR spectroscopy (reductions in N-Acetylaspartate) and markers preceding dementia in humans (reduced amyloid ?(A?42)/tau ratio in CSF); and (c) markers of neuroinflammation and neurotoxicity (CSF levels of kynurenine pathway metabolites). Aim 2 will test whether ELA accelerates age- related deficits in stress/emotional regulation mediated by PFC-amygdala circuits (HPA axis, Human Intruder and dot-probe tasks), and cognition: attention (continuous performance task), executive function and cognitive flexibility mediated by PFC circuits (Intradimensional/Extradimensional discrimination), and spatial relational memory mediated by HIPP-PFC circuits (spatial memory span). Aim 3 will examine associations between longitudinal trajectories of neural measures (Aim 1) and cognitive outcomes (Aim 2); we will use innovative longitudinal statistical approaches developing individualized trajectory biomarkers of risk and resilience that move beyond association to establishing statistical causation. The hypothesis is that the ELA group will show early biomarkers of stress and accelerated trajectories of biological and neurocognitive aging in middle age.