Heightened extended amygdala metabolism following threat characterizes the early phenotypic risk to develop anxiety-related psychopathology.

Children with an anxious temperament are prone to heightened shyness and behavioral inhibition (BI). When chronic and extreme, this anxious, inhibited phenotype is an important early-life risk factor for the development of anxiety disorders, depression and co-morbid substance abuse. Individuals with extreme anxious temperament often show persistent distress in the absence of immediate threat and this contextually inappropriate anxiety predicts future symptom development. Despite its clear clinical relevance, the neural circuitry governing the maladaptive persistence of anxiety remains unclear. Here, we used a well-established nonhuman primate model of childhood temperament and high-resolution 18fluorodeoxyglucose positron emission tomography (FDG-PET) imaging to understand the neural systems governing persistent anxiety and to clarify their relevance to early-life phenotypic risk. We focused on BI, a core component of anxious temperament, because it affords the moment-by-moment temporal resolution needed to assess contextually appropriate and inappropriate anxiety. From a pool of 109 peri-adolescent rhesus monkeys, we formed groups characterized by high or low levels of BI, as indexed by freezing in response to an unfamiliar human intruder's profile. The high-BI group showed consistently elevated signs of anxiety and wariness across >2 years of assessments. At the time of brain imaging, 1.5 years after initial phenotyping, the high-BI group showed persistently elevated freezing during a 30-min 'recovery' period following an encounter with the intruder-more than an order of magnitude greater than the low-BI group-and this was associated with increased metabolism in the bed nucleus of the stria terminalis, a key component of the central extended amygdala. These observations provide a neurobiological framework for understanding the early phenotypic risk to develop anxiety-related psychopathology, for accelerating the development of improved interventions, and for understanding the origins of childhood temperament.

Awareness of Emotional Stimuli Determines the Behavioral Consequences of Amygdala Activation and Amygdala-Prefrontal Connectivity.

Conscious awareness of negative cues is thought to enhance emotion-regulatory capacity, but the neural mechanisms underlying this effect are unknown. Using continuous flash suppression (CFS) in the MRI scanner, we manipulated visual awareness of fearful faces during an affect misattribution paradigm, in which preferences for neutral objects can be biased by the valence of a previously presented stimulus. The amygdala responded to fearful faces independently of awareness. However, when awareness of fearful faces was prevented, individuals with greater amygdala responses displayed a negative bias toward unrelated novel neutral faces. In contrast, during the aware condition, inverse coupling between the amygdala and prefrontal cortex reduced this bias, particularly among individuals with higher structural connectivity in the major white matter pathway connecting the prefrontal cortex and amygdala. Collectively, these results indicate that awareness promotes the function of a critical emotion-regulatory network targeting the amygdala, providing a mechanistic account for the role of awareness in emotion regulation.

Evolutionarily conserved prefrontal-amygdalar dysfunction in early-life anxiety.

Some individuals are endowed with a biology that renders them more reactive to novelty and potential threat. When extreme, this anxious temperament (AT) confers elevated risk for the development of anxiety, depression and substance abuse. These disorders are highly prevalent, debilitating and can be challenging to treat. The high-risk AT phenotype is expressed similarly in children and young monkeys and mechanistic work demonstrates that the central (Ce) nucleus of the amygdala is an important substrate. Although it is widely believed that the flow of information across the structural network connecting the Ce nucleus to other brain regions underlies primates' capacity for flexibly regulating anxiety, the functional architecture of this network has remained poorly understood. Here we used functional magnetic resonance imaging (fMRI) in anesthetized young monkeys and quietly resting children with anxiety disorders to identify an evolutionarily conserved pattern of functional connectivity relevant to early-life anxiety. Across primate species and levels of awareness, reduced functional connectivity between the dorsolateral prefrontal cortex, a region thought to play a central role in the control of cognition and emotion, and the Ce nucleus was associated with increased anxiety assessed outside the scanner. Importantly, high-resolution 18-fluorodeoxyglucose positron emission tomography imaging provided evidence that elevated Ce nucleus metabolism statistically mediates the association between prefrontal-amygdalar connectivity and elevated anxiety. These results provide new clues about the brain network underlying extreme early-life anxiety and set the stage for mechanistic work aimed at developing improved interventions for pediatric anxiety.

CRHR1 genotypes, neural circuits and the diathesis for anxiety and depression.

The corticotrophin-releasing hormone (CRH) system integrates the stress response and is associated with stress-related psychopathology. Previous reports have identified interactions between childhood trauma and sequence variation in the CRH receptor 1 gene (CRHR1) that increase risk for affective disorders. However, the underlying mechanisms that connect variation in CRHR1 to psychopathology are unknown. To explore potential mechanisms, we used a validated rhesus macaque model to investigate association between genetic variation in CRHR1, anxious temperament (AT) and brain metabolic activity. In young rhesus monkeys, AT is analogous to the childhood risk phenotype that predicts the development of human anxiety and depressive disorders. Regional brain metabolism was assessed with (18)F-labeled fluoro-2-deoxyglucose (FDG) positron emission tomography in 236 young, normally reared macaques that were also characterized for AT. We show that single nucleotide polymorphisms (SNPs) affecting exon 6 of CRHR1 influence both AT and metabolic activity in the anterior hippocampus and amygdala, components of the neural circuit underlying AT. We also find evidence for association between SNPs in CRHR1 and metabolism in the intraparietal sulcus and precuneus. These translational data suggest that genetic variation in CRHR1 affects the risk for affective disorders by influencing the function of the neural circuit underlying AT and that differences in gene expression or the protein sequence involving exon 6 may be important. These results suggest that variation in CRHR1 may influence brain function before any childhood adversity and may be a diathesis for the interaction between CRHR1 genotypes and childhood trauma reported to affect human psychopathology.

Serotonin transporter binding and genotype in the nonhuman primate brain using [C-11]DASB PET.

UNLABELLED: The length polymorphism of the serotonin (5-HT) transporter gene promoter region has been implicated in altered 5-HT function and, in turn, neuropsychiatric illnesses, such as anxiety and depression. The nonhuman primate has been used as a model to study anxiety-related mechanisms in humans based upon similarities in behavior and the presence of a similar 5-HT transporter gene polymorphism. Stressful and threatening contexts in the nonhuman primate model have revealed 5-HT transporter genotype dependent differences in regional glucose metabolism. Using the rhesus monkey, we examined the extent to which serotonin transporter genotype is associated with 5-HT transporter binding in brain regions implicated in emotion-related pathology. METHODS: Genotype data and high resolution PET scans were acquired in 29 rhesus (Macaca mulatta) monkeys. [C-11]DASB dynamic PET scans were acquired for 90 min in the anesthetized animals and images of distribution volume ratio (DVR) were created to serve as a metric of 5-HT transporter binding for group comparison based on a reference region method of analysis. Regional and voxelwise statistical analysis were performed with corrections for anatomical differences in gray matter probability, sex, age and radioligand mass. RESULTS: There were no significant differences when comparing l/l homozygotes with s-carriers in the regions of the brain implicated in anxiety and mood related illnesses (amygdala, striatum, thalamus, raphe nuclei, temporal and prefrontal cortex). There was a significant sex difference in 5-HT transporter binding in all regions with females having 18%-28% higher DVR than males. CONCLUSIONS: Because these findings are consistent with similar genotype findings in humans, this further strengthens the use of the rhesus model for studying anxiety-related neuropathologies.

Differential behavioral state-dependence in the burst properties of CA3 and CA1 neurons.

Brief bursts of fast high-frequency action potentials are a signature characteristic of CA3 and CA1 pyramidal neurons. Understanding the factors determining burst and single spiking is potentially significant for sensory representation, synaptic plasticity and epileptogenesis. A variety of models suggest distinct functional roles for burst discharge, and for specific characteristics of the burst in neural coding. However, little in vivo data demonstrate how often and under what conditions CA3 and CA1 actually exhibit burst and single spike discharges. The present study examined burst discharge and single spiking of CA3 and CA1 neurons across distinct behavioral states (awake-immobility and maze-running) in rats. In both CA3 and CA1 spike bursts accounted for less than 20% of all spike events. CA3 neurons exhibited more spikes per burst, greater spike frequency, larger amplitude spikes and more spike amplitude attenuation than CA1 neurons. A major finding of the present study is that the propensity of CA1 neurons to burst was affected by behavioral state, while the propensity of CA3 to burst was not. CA1 neurons exhibited fewer bursts during maze running compared with awake-immobility. In contrast, there were no differences in burst discharge of CA3 neurons. Neurons in both subregions exhibited smaller spike amplitude, fewer spikes per burst, longer inter-spike intervals and greater spike amplitude attenuation within a burst during awake-immobility compared with maze running. These findings demonstrate that the CA1 network is under greater behavioral state-dependent regulation than CA3. The present findings should inform both theoretic and computational models of CA3 and CA1 function.

Hippocampal and amygdalar involvement in discriminatory place learning.

A conflict task was developed that simultaneously examines place aversion learning and fear-motivated context discrimination. The task superimposed Pavlovian discriminative fear conditioning on an appetitively motivated instrumental response (alternation). Rats were trained to alternate along a high-walled, diamond-shaped runway between two chambers for food. On half of the trials, a tone CS signaled the fact that a fixed section at the apex of the runway was electrified. Both the tone and the shock were turned on at the beginning of, and remained on for the duration of, each tone trial. A new trial was initiated at the time the animal entered the subsequent food chamber. Therefore, during a tone trial, in order to attain additional food reinforcement, the animal had to cross over the electrified region at the runway apex. Behavioral performance of rats with small lesions of the amygdala or dorsal hippocampus (DH) was compared with that of sham-operated controls. All groups displayed significant discriminative responding, hesitating more on tone trials while in areas of the runway adjacent to the shock region. Animals with lesions of the DH were similar to controls with respect to the tone-mediated discrimination, yet were delayed in the initial expression of a location-specific fear response. Conversely, amygdala lesions did not affect place learning; however, these animals were impaired in their suppression of the fear response following repeated unpaired trials.

Age-related deficits in the ability to encode contextual change: a place cell analysis.

Aging is known to impair the formation of episodic memory, a process dependent upon the integrity of the hippocampal region. To investigate this issue, hippocampal place cells were recorded from middle-aged and old F-344 male rats while running on a "figure-8" track. The top and bottom arcs of the track were removed, converting it into a plus maze, and the animals were required to conduct a working memory task. Following this change in task, the arcs were replaced and the animals again ran the figure-8 task. Analysis of place fields across the recording session demonstrated that both middle-aged and old rats had reliable representations of the figure-8 task. A comparison of place fields between different behavioral tasks (figure-8 and plus maze) demonstrated a change in the hippocampal representation of the environment in both age groups, despite the fact that the animals remained on the maze throughout the recording session. Notably, place cells in old animals were less affected by the change in task than those in middle-aged animals. The results suggest that hippocampal neurons reflect significant behavioral events within a given environment. Furthermore, the data indicate that age-related episodic memory deficits may result from decreased sensitivity of the hippocampal network to respond to meaningful changes in the environment.

Hippocampal dysfunction during aging I: deficits in memory consolidation.

Numerous ablation studies indicate a critical role for the hippocampal system in establishing or consolidating certain types of memory. Normal aging manifests by selective neurobiological changes in the hippocampal formation and on performance of tasks that require a functional hippocampus, including retention of contextual fear conditioning. To determine if impairments in the consolidation process contribute to memory dysfunction in aging, middle-aged and aged rats were fear conditioned and subsequently received dorsal hippocampal lesions or sham surgery after a 1, 7, 14, or 28-day interval. During retention tests, middle-aged rats exhibited a temporally graded retrograde amnesia of contextual fear conditioning, whereas aged rats manifested contextual memory impairments at all intervals. We postulate that the lack of consolidation in aged animals relates to previous findings of age-related changes in neuroanatomy and neurophysiological plasticity. The present findings suggest that impaired hippocampal consolidation contributes to age-related learning and memory deficits.

Age-related deficits on the radial maze and in fear conditioning: hippocampal processing and consolidation.

Young adult, middle-aged, and old male F-344 rats were assessed for their hippocampal ability. This was accomplished by examining the animals on two different paradigms, each incorporating a simultaneous measure of hippocampal-dependent and -independent processing. The animals were fear conditioned and then tested for retention of the conditioning context and tone. This was followed by an 8-arm radial maze task which combined spatial working and cued reference memory elements. The two paradigms are compared in terms of task demands, potential confounds, and validity for aging studies. The results indicate that the performance of the animals on the two tasks is correlated. Age-related deficits limited to the hippocampal aspects of the above tasks were found, with no deficits found in the analogous but hippocampus-independent aspects of these tasks. The function of the hippocampus in incorporating new memories is time-related. Therefore, the possibility of age-related changes in consolidation was examined. It has previously been shown on the fear conditioning paradigm that the hippocampus is involved in retention of the aversive context for approximately 28 days. In the present study, an attempt was made to test the animals for retention of the conditioning context both early into the period of consolidation (10 days) and after consolidation should have been completed (52 days). The results indicate that, initially, the old animals show comparable retention to young rats. When examined later, young animals showed a stronger retention of the conditioning context than they had previously. The aged rats, however, did not seem to benefit from this additional period of time and in fact showed a decrease in retention of the conditioning context. The data are interpreted in terms of consolidation, alternative explanations of the data are presented, and suggestions are given for future research. Finally, the implications of such age-related changes in hippocampal consolidation on learning and memory are discussed.