Central amygdala nucleus (Ce) gene expression linked to increased trait-like Ce metabolism and anxious temperament in young primates.
Journal: 2013/January - Proceedings of the National Academy of Sciences of the United States of America
ISSN: 1091-6490
Abstract:
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.
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Proc Natl Acad Sci U S A 109(44): 18108-18113

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

Supplementary Material

Supporting Information:
Departments of Psychology and
Psychiatry and
HealthEmotions Research Institute, University of Wisconsin, Madison, WI, 53719; and
Waisman Laboratory for Brain Imaging and Behavior, University of Wisconsin, Madison, WI, 53705
To whom correspondence may be addressed. E-mail: ude.csiw@xofsa or ude.csiw@nilakn.
Edited by Marcus E. Raichle, Washington University in St. Louis, St. Louis, MO, and approved September 11, 2012 (received for review April 23, 2012)

Author contributions: S.E.S., R.J.D., P.H.R., and N.H.K. designed research; A.S.F., S.E.S., S.A.N., P.H.R., and N.H.K. performed research; A.S.F. contributed new reagents/analytic tools; A.S.F., J.A.O., P.H.R., and N.H.K. analyzed data; and A.S.F., J.A.O., and N.H.K. wrote the paper.

Edited by Marcus E. Raichle, Washington University in St. Louis, St. Louis, MO, and approved September 11, 2012 (received for review April 23, 2012)

Abstract

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.

Keywords: positron-emission tomography, microarray, brain imaging
Abstract

The ability to identify brain mechanisms underlying the risk during childhood for developing anxiety and depression is critical for establishing novel early-life interventions aimed at preventing the chronic and debilitating outcomes associated with these common illnesses. To this end, we have optimized a model of anxious temperament (AT), the conserved at-risk phenotype, in young developing rhesus monkeys (14). The rhesus monkey is ideal for studying the origin of human AT because these species share the genetic, neural, and phenotypic underpinnings of complex social and emotional functioning (510). Importantly, the rhesus developmental model bridges the critical gap between human psychopathology and rodent models, allowing for translation to humans by using in vivo imaging measures and translation to rodents by using ex vivo molecular methods. Thus, the unique hypotheses that can be generated from the rhesus model are invaluable in guiding both imaging studies in children and mechanistic efforts in rodents.

Of particular relevance to the AT rhesus model is the relatively recent evolutionary divergence between rhesus monkeys and humans (25 million years) compared with rodents and humans (70 million years) (5). This evolutionary closeness is reflected in the species’ similarities in social and emotional behaviors. These homologies, instantiated in their conserved genetic and neural systems, underlie the ability of both humans and rhesus monkeys to form and maintain the relationships necessary for living in complex social environments. In this regard, the experience of anxiety has evolved in primates to motivate the formation of long-lasting attachment bonds that serve to increase security and group cohesion. The comparable rearing practices shared by these species (e.g., close mother–infant bonding) promote early social/emotional learning, which serves to adaptively regulate anxiety and promote survival (7).

Although periods of marked anxiety and fear are common during early childhood, most children overcome these anxieties through learning associated with experience and maturation. As they develop, typical children learn to discern real threats from distorted fears and, in concert, effectively regulate their behavior to adaptively cope. However, a subset of children with extreme AT do not develop this capacity, maintaining a stable anxious disposition that confers increased risk for the development of anxiety and mood disorders (1113). AT begins as early shyness and is later characterized by chronic anxiety, negative affect, and worry (14). AT is also associated with increased activity of stress-sensitive peripheral systems, including increased pituitary–adrenal tone and heightened sympathetic activity (11).

Because early social-emotional learning is critical for the adaptive regulation of anxiety, we have been especially interested in processes demonstrated to underlie learning during development. Furthermore, recent preclinical and clinical research has identified neurotrophic factors (15) and other neuroplastic processes as critical for overcoming adult psychopathology (1618). Therefore, we hypothesized that altered neurotrophic processes in the young brain would lead to the emergence and maintenance of childhood AT. In particular, we theorize that deficits in the ability to modify the connections and composition of AT’s neural substrate could result in a failure to learn how to adaptively regulate anxiety, which can manifest as a tendency to generalize perceptions of threat to neutral stimuli. Because AT can be identified early in life, characterizing the biological factors that promote the maintenance of stable AT can potentially lead to targeted early-life interventions aimed at decreasing the risk for developing psychopathology.

Similar to anxious children, young monkeys with high levels of AT are those that show increased freezing, decreased vocalizations, and increased cortisol when exposed to the no-eye contact condition (NEC) of the human-intruder paradigm, an ethologically relevant mild social threat. Our studies demonstrated that, like human AT, monkey AT is trait-like and heritable (19). Using functional brain imaging in conjunction with ex vivo molecular analyses of relevant brain regions, the monkey model allows for the longitudinal study of AT and its underlying neural substrates. With functional brain imaging, we identified the central nucleus of the amygdala (Ce) and anterior hippocampus as components of the neural circuit underlying AT (2, 19). Moreover, we found that young primates with high AT have increased metabolism in these regions when studied in both stressful and nonstressful contexts (2). These data set the stage for in-depth molecular studies in primates focused on understanding the mechanisms mediating the function of the brain regions underlying AT.

The Ce is of interest because its efferent projections coordinate autonomic, hormonal, behavioral, and emotional responses to stress (20), and Ce lesions in monkeys are sufficient to reduce AT (21). Furthermore, rodent studies demonstrate that direct Ce manipulations markedly alter unconditioned anxiety responses (22), similar to those elicited by novel or potentially threatening situations in children with high AT. The prefrontal cortex and other amygdala nuclei primarily influence fear and anxiety-related responding via the Ce, where intra-Ce microcircuits play a critical role dynamically gating these inputs (2326). Recent work in rhesus monkeys demonstrates that, unlike most amygdala nuclei, the Ce continues to mature from the first year of life into early adulthood (27). This protracted developmental period suggests that Ce maturation may be particularly susceptible to environmental influences. A causal relation between social group size and dorsal amygdala volume demonstrates the importance of social influences on the primate amygdala (28). These findings are consistent with our data highlighting the importance of environmental contributions to Ce metabolism as it relates to early-life AT (19). For these reasons, we selected the Ce for in depth molecular analyses, with a particular focus on processes within the Ce that underlie learning. Although learning-related research has generally focused on the hippocampus (e.g., refs. 29 and 30) and basal/lateral amygdala regions (31), recent rodent studies highlight the role of plasticity and emotional learning in the Ce (32, 33). In addition to its role in anxiety, the Ce has recently been linked to habit formation (34) and, at a cellular and neurochemical level, it has much in common with the striatum (35), a structure known to mediate the development of long-term ingrained response patterns (36). Although Ce microcircuits are ideally suited to perform the childhood learning that results in adaptive anxiety, when Ce learning is disrupted, it could result in trait-like habitual fear and anxiety responding.

Building on our finding that individual differences in Ce metabolism predict AT, we performed mRNA expression studies in Ce tissue collected from young monkeys repeatedly phenotyped for AT and its associated brain metabolism. This unique, multilevel approach combines the power of functional brain imaging with the potential of gene expression studies to characterize the mRNAs that could underlie the risk for developing anxiety and depression. We hypothesized that high-AT individuals would have alterations in mRNAs within the Ce that reflect the influences of experience on the persistent expression of anxiety. Specifically, we predicted a role for mRNAs encoding molecules with the potential to facilitate habitual anxiety and developmentally appropriate adaptive fear learning. Such alterations are of particular interest, because manipulations of these substrates could result in treatments for high-AT children that would facilitate their ability to modify and adaptively regulate their anxiety.

Accordingly, a subset of 24 animals was selected from 238 rhesus monkeys that were initially characterized for behavior and brain metabolism. The 238 monkeys were injected with [F]-fluoro-2-deoxyglucose (FDG) and exposed for 30 min to the NEC condition that elicits the AT phenotype (19). During NEC, a human (“the intruder”) enters the test room and presents her profile to the monkey, avoiding eye contact (1). Following NEC exposure, animals were anesthetized, and high-resolution positron-emission tomography (PET) scans were performed to examine the integrated brain metabolism that occurred during the preceding 30-min NEC exposure. FDG-PET is optimal for simultaneously assessing sustained neural activity and natural behavior in unconstrained individuals because FDG is taken up into metabolically active cells over the course of ∼30 min and remains trapped for the duration of its ∼110-min half-life. This extended assessment of brain metabolism is ideal for studying the neural underpinnings of AT, which are sustained over time.

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Acknowledgments

We thank the staff at the Wisconsin National Primate Center, the Harlow Center for Biological Psychology, the HealthEmotions Research Institute, and The Waisman Laboratory for Brain Imaging and Behavior for facilitating our research. We also thank A. Shackman, A. Alexander, B. Christian, L. Ahlers, A. Converse, T. Oakes, H. Van Valkenberg, K. Myer, J. Spears, E. Larson, and D. French for their technical and analytic assistance in running subjects and analyzing data. This work was supported by National Institutes of Health (NIH) Grants MH91550, MH046729, MH081884, MH084051, HD008352, and HD003352; NIH Training Grant MH018931; the Wisconsin National Primate Research Center (through NIH Grants P51OD011106 and P51RR000167); and the HealthEmotions Research Institute.

Acknowledgments

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1206723109/-/DCSupplemental.

Footnotes

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