Differential Effects of the Inactivation of Anterior and Posterior Orbitofrontal Cortex on Affective Responses to Proximal and Distal Threat, and Reward Anticipation in the Common Marmoset.

Structural and functional abnormalities of the orbitofrontal cortex (OFC) have been implicated in affective disorders that manifest anxiety-related symptoms. However, research into the functions of primate OFC has predominantly focused on reward-oriented rather than threat-oriented responses. To redress this imbalance, the present study performed a comprehensive analysis of the independent role of 2 distinct subregions of the central OFC (anterior area 11; aOFC and posterior area 13; pOFC) in the processing of distal and proximal threat. Temporary inactivation of both aOFC and pOFC heightened responses to distal threat in the form of an unknown human, but not to proximal threat assessed in a discriminative Pavlovian conditioning task. Inactivation of the aOFC, however, did unexpectedly blunt conditioned threat responses, although the effect was not valence-specific, as conditioned appetitive responses were similarly blunted and appeared restricted to a discriminative version of the task (when both CS- and CS+ are present within a session). Inactivation of the pOFC did not affect conditioned responses to either proximal threat or reward and basal cardiovascular activity was unaffected by manipulations of activity in either subregion. The results highlight the contribution of aOFC and pOFC to regulation of responses to more distal uncertain but not proximal, certain threat and reveal their opposing contribution to that of the immediately adjacent medial OFC, area 14.

Ventromedial prefrontal area 14 provides opposing regulation of threat and reward-elicited responses in the common marmoset.

The ventromedial prefrontal cortex (vmPFC) is a key brain structure implicated in mood and anxiety disorders, based primarily on evidence from correlational neuroimaging studies. Composed of a number of brain regions with distinct architecture and connectivity, dissecting its functional heterogeneity will provide key insights into the symptomatology of these disorders. Focusing on area 14, lying on the medial and orbital surfaces of the gyrus rectus, this study addresses a key question of causality. Do changes in area 14 activity induce changes in threat- and reward-elicited responses within the nonhuman primate, the common marmoset, similar to that seen in mood and anxiety disorders? Area 14 overactivation was found to induce heightened responsivity to uncertain, low-imminence threat while blunting cardiovascular and behavioral anticipatory arousal to high-value food reward. Conversely, inactivation enhanced the arousal to high-value reward cues while dampening the acquisition of cardiovascular and behavioral responses to a Pavlovian threat cue. Basal cardiovascular activity, including heart rate variability and sympathovagal balance, which are dysfunctional in mood and anxiety disorders, are insensitive to alterations in area 14 activity as is the extinction of conditioned threat responses. The distinct pattern of dysregulation compared to neighboring region area 25 highlights the heterogeneity of function within vmPFC and reveals how the effects of area 14 overactivation on positive and negative reactivity mirror symptoms of anhedonia and anxiety that are so often comorbid in mood disorders.

Differential Contribution of Anterior and Posterior Midcingulate Subregions to Distal and Proximal Threat Reactivity in Marmosets.

The midcingulate cortex (MCC) is associated with cognition and emotion regulation. Structural and correlational functional evidence suggests that rather than being homogenous, the MCC may have dissociable functions that can be mapped onto distinct subregions. In this study, we use the marmoset monkey to causally investigate the contributions of two proposed subregions of the MCC: the anterior and posterior midcingulate cortices (aMCC and pMCC) to behavioral and cardiovascular correlates of threat processing relevant to anxiety disorders. Transient inactivation of the aMCC decreased anxiety-like responses to a postencounter distal threat, namely an unfamiliar human intruder, while inactivation of the pMCC showed a mild but opposing effect. Furthermore, although inactivation of neither MCC subregions had any effect on basal cardiovascular activity, aMCC inactivation blunted the expression of both cardiovascular and behavioral conditioned responses to a predictable proximal threat (a rubber snake) during the extinction in a Pavlovian conditioning task, with pMCC inactivation having again an opposing effect, but primarily on the behavioral response. These findings suggest that the MCC is indeed functionally heterogeneous with regards to its role in threat processing, with aMCC providing a marked facilitative contribution to the expression of the emotional response to both proximal and distal threat.

Glutamate Within the Marmoset Anterior Hippocampus Interacts with Area 25 to Regulate the Behavioral and Cardiovascular Correlates of High-Trait Anxiety.

High-trait anxiety is a risk factor for the development of affective disorders and has been associated with decreased cardiovascular and behavioral responsivity to acute stressors in humans that may increase the risk of developing cardiovascular disease. Although human neuroimaging studies of high-trait anxiety reveals dysregulation in primate cingulate areas 25 and 32 and the anterior hippocampus (aHipp) and rodent studies reveal the importance of aHipp glutamatergic hypofunction, the causal involvement of aHipp glutamate and its interaction with these areas in the primate brain is unknown. Accordingly, we correlated marmoset trait anxiety scores to their postmortem aHipp glutamate levels and showed that low glutamate in the right aHipp is associated with high-trait anxiety in marmosets. Moreover, pharmacologically increasing aHipp glutamate reduced anxiety levels in highly anxious marmosets in two uncertainty-based tests of anxiety: exposure to a human intruder with uncertain intent and unpredictable loud noise. In the human intruder test, increasing aHipp glutamate decreased anxiety by increasing approach to the intruder. In the unpredictable threat test, animals showed blunted behavioral and cardiovascular responsivity after control infusions, which was normalized by increasing aHipp glutamate. However, this aHipp-mediated anxiolytic effect was blocked by simultaneous pharmacological inactivation of area 25, but not area 32, areas which when inactivated independently reduced and had no effect on anxiety, respectively. These findings provide causal evidence in male and female primates that aHipp glutamatergic hypofunction and its regulation by area 25 contribute to the behavioral and cardiovascular symptoms of endogenous high-trait anxiety.SIGNIFICANCE STATEMENT High-trait anxiety predisposes sufferers to the development of anxiety and depression. Although neuroimaging of these disorders and rodent modeling implicate dysregulation in hippocampal glutamate and the subgenual/perigenual cingulate cortices (areas 25/32), the causal involvement of these structures in endogenous high-trait anxiety and their interaction are unknown. Here, we demonstrate that increased trait anxiety in marmoset monkeys correlates with reduced hippocampal glutamate and that increasing hippocampal glutamate release in high-trait-anxious monkeys normalizes the aberrant behavioral and cardiovascular responsivity to potential threats. This normalization was blocked by simultaneous inactivation of area 25, but not area 32. These findings provide casual evidence in primates that hippocampal glutamatergic hypofunction regulates endogenous high-trait anxiety and the hippocampal-area 25 circuit is a potential therapeutic target.

Hippocampal Interaction With Area 25, but not Area 32, Regulates Marmoset Approach-Avoidance Behavior.

Affective disorders are associated with increased sensitivity to negative feedback that influences approach-avoidance decision making. Although neuroimaging studies of these disorders reveal dysregulation in primate cingulate areas 25 and 32 and the anterior hippocampus (aHipp), the causal involvement of these structures and their interaction in the primate brain is unknown. We therefore investigated the effects of localized pharmacological manipulations of areas 25 and 32 and/or the aHipp of the marmoset monkey on performance of an anxiolytic-sensitive instrumental decision-making task in which an approach-avoidance conflict is created by pairing a response with reward and punishment. During control infusions animals avoided punishment, but this bias was reduced by increasing glutamate release within the aHipp or area 32, and inactivation or 5-HT1a antagonism within area 25. Conversely, increasing glutamate release in area 25 enhanced punishment avoidance but, in contrast to previous reports, area 32 and aHipp inactivations had no effect. Simultaneous inactivation or 5-HT1a antagonism within area 25, but not area 32, abolished the reduced punishment avoidance seen after increasing aHipp glutamate. Besides providing causal evidence that these primate areas differentially regulate negative feedback sensitivity, this study links the decision-making deficits in affective disorders to aberrant aHipp-area 25 circuit activity.

Selective Role of the Putamen in Serial Reversal Learning in the Marmoset.

Fronto-striatal circuitry involving the orbitofrontal cortex has been identified as mediating successful reversal of stimulus-outcome contingencies. The region of the striatum that most contributes to reversal learning remains unclear, with studies in primates implicating both caudate nucleus and putamen. We trained four marmosets on a touchscreen-based serial reversal task and implanted each with cannulae targeting both putamen and caudate bilaterally. This allowed reversible inactivation of the two areas within the same monkeys, but across separate sessions, to directly investigate their respective contributions to reversal performance. Behavioral sensitivity to the GABAA agonist muscimol varied across subjects and between brain regions, so each marmoset received a range of doses. Intermediate doses of intra-putamen muscimol selectively impaired reversal performance, leaving the baseline discrimination phase unchanged. There was no effect of low doses and high doses were generally disruptive. By contrast, low doses of intra-caudate muscimol improved reversal performance, while high doses impaired both reversal and baseline discrimination performance. These data provide evidence for a specific role of the putamen in serial reversal learning, which may reflect the more habitual nature of repeated reversals using the same stimulus pair.

Role of Central Serotonin in Anticipation of Rewarding and Punishing Outcomes: Effects of Selective Amygdala or Orbitofrontal 5-HT Depletion.

Understanding the role of serotonin (or 5-hydroxytryptamine, 5-HT) in aversive processing has been hampered by the contradictory findings, across studies, of increased sensitivity to punishment in terms of subsequent response choice but decreased sensitivity to punishment-induced response suppression following gross depletion of central 5-HT. To address this apparent discrepancy, the present study determined whether both effects could be found in the same animals by performing localized 5-HT depletions in the amygdala or orbitofrontal cortex (OFC) of a New World monkey, the common marmoset. 5-HT depletion in the amygdala impaired response choice on a probabilistic visual discrimination task by increasing the effectiveness of misleading, or false, punishment and reward, and decreased response suppression in a variable interval test of punishment sensitivity that employed the same reward and punisher. 5-HT depletion in the OFC also disrupted probabilistic discrimination learning and decreased response suppression. Computational modeling of behavior on the discrimination task showed that the lesions reduced reinforcement sensitivity. A novel, unitary account of the findings in terms of the causal role of 5-HT in the anticipation of both negative and positive motivational outcomes is proposed and discussed in relation to current theories of 5-HT function and our understanding of mood and anxiety disorders.

Chemogenetics identifies separate area 25 brain circuits involved in anhedonia and anxiety in marmosets.

Poor outcomes are common in individuals with anxiety and depression, and the brain circuits underlying symptoms and treatment responses remain elusive. To elucidate these neural circuits, experimental studies must specifically manipulate them, which is only possible in animals. Here, we used a chemogenetics strategy involving engineered designer receptors exclusively activated by designer drugs (DREADDs) to activate a region of the marmoset brain that is dysfunctional in human patients with major depressive disorder, called the subcallosal anterior cingulate cortex area 25 (scACC-25). Using this DREADDs system, we identified separate scACC-25 neural circuits that underlie specific components of anhedonia and anxiety in marmosets. Activation of the neural pathway connecting the scACC-25 to the nucleus accumbens (NAc) caused blunting of anticipatory arousal (a form of anhedonia) in marmosets in response to a reward-associated conditioned stimulus in an appetitive Pavlovian discrimination test. Separately, activation of the circuit between the scACC-25 and the amygdala increased a measure of anxiety (the threat response score) when marmosets were presented with an uncertain threat (human intruder test). Using the anhedonia data, we then showed that the fast-acting antidepressant ketamine when infused into the NAc of marmosets prevented anhedonia after scACC-25 activation for more than 1 week. These neurobiological findings provide targets that could contribute to the development of new treatment strategies.

Serotonin at the level of the amygdala and orbitofrontal cortex modulates distinct aspects of positive emotion in primates.

Impaired top-down regulation of the amygdala, and its modulation by serotonin (5-HT), is strongly implicated in the dysregulation of negative emotion that characterizes a number of affective disorders. However, the contribution of these mechanisms to the regulation of positive emotion is not well understood. This study investigated the role of 5-HT within the amygdala and the orbitofrontal cortex (OFC), on the expression of appetitive Pavlovian conditioned emotional responses and their reversal in a primate, the common marmoset. Its effects were compared to those of the amygdala itself. Having developed conditioned autonomic and behavioural responses to an appetitive cue prior to surgery, marmosets with excitotoxic amygdala lesions failed to display such conditioned autonomic arousal at retention, but still displayed intact cue-directed conditioned behaviours. In contrast, 5,7-DHT infusions into the amygdala, reducing extracellular 5-HT levels, selectively enhanced the expression of appetitive conditioned behaviour at retention. Similar infusions into the OFC, producing marked reductions in post-mortem 5-HT tissue levels, had no overall effect on autonomic or behavioural responses, either at retention or during reversal learning, but caused an uncoupling of these responses, thereby fractionating emotional output. These data demonstrate the critical role of the amygdala in the expression of appetitive autonomic conditioning, and the region-selective contribution of 5-HT in the amygdala and OFC, respectively, to the expression of conditioned behaviour and the overall coordination of the emotional response. They provide insight into the neurochemical mechanisms underlying the regulation of positive emotional responses, advancing our understanding of the neural basis of pathologically dysregulated emotion.

Avoidant Coping Style to High Imminence Threat Is Linked to Higher Anxiety-Like Behavior.

Human studies with self-reported measures have suggested a link between an avoidant coping style and high anxiety. Here, using the common marmoset as a model, we characterize the latent factors underlying behavioral responses of these monkeys towards low and high imminence threat and investigate if a predominantly avoidant behavioral response to high imminence threat is associated with greater anxiety-like behavior in a context of low imminence threat. Exploratory factor analysis (EFA) of the human intruder test of low imminence threat revealed a single factor in which a combination of active vigilance and avoidance responses underpinned anxiety-like behavior. In contrast, two negatively-associated factors were revealed in the model snake test reflecting active and avoidant coping to high imminence threat. Subsequent analysis showed that animals with a predominantly avoidant coping style on the model snake test displayed higher anxiety-like behavior on the human intruder test, findings consistent with those described in humans. Together they illustrate the richness of the behavioral repertoire displayed by marmosets in low and high imminence threatening contexts and the additional insight that factor analysis can provide by identifying the latent factors underlying these complex behavioral datasets. They also highlight the translational value of this approach when studying the neural circuits underlying complex anxiety-like states in this primate model.

Fractionating Blunted Reward Processing Characteristic of Anhedonia by Over-Activating Primate Subgenual Anterior Cingulate Cortex.

Anhedonia is a core symptom of depression, but the underlying neurobiological mechanisms are unknown. Correlative neuroimaging studies implicate dysfunction within ventromedial prefrontal cortex, but the causal roles of specific subregions remain unidentified. We addressed these issues by combining intracerebral microinfusions with cardiovascular and behavioral monitoring in marmoset monkeys to show that over-activation of primate subgenual anterior cingulate cortex (sgACC, area 25) blunts appetitive anticipatory, but not consummatory, arousal, whereas manipulations of adjacent perigenual ACC (pgACC, area 32) have no effect. sgACC/25 over-activation also reduces the willingness to work for reward. 18F-FDG PET imaging reveals over-activation induced metabolic changes in circuits involved in reward processing and interoception. Ketamine treatment ameliorates the blunted anticipatory arousal and reverses associated metabolic changes. These results demonstrate a causal role for primate sgACC/25 over-activity in selective aspects of impaired reward processing translationally relevant to anhedonia, and ketamine's modulation of an affective network to exert its action.

Converging Prefronto-Insula-Amygdala Pathways in Negative Emotion Regulation in Marmoset Monkeys.

BACKGROUND: Impaired regulation of emotional responses to potential threat is a core feature of affective disorders. However, while the subcortical circuitry responsible for processing and expression of fear has been well characterized, the top-down control of this circuitry is less well understood. Our recent studies demonstrated that heightened emotionality, as measured both physiologically and behaviorally, during conditioned fear and innate/social threat was induced, independently, by excitotoxic lesions of either the anterior orbitofrontal cortex (antOFC) or ventrolateral prefrontal cortex (vlPFC). An important outstanding question is whether the antOFC and vlPFC act on common or distinct downstream targets to regulate negative emotion. METHODS: The question was addressed by combining localized excitotoxic lesions in the PFC of a nonhuman primate and functional neuroimaging ([18F]fluorodeoxyglucose positron emission tomography) with a fear-regulating extinction paradigm. Marmoset monkeys with unilateral lesions of either the antOFC or vlPFC were scanned immediately following exposure to a fearful or safe context, and differences in [18F]fluorodeoxyglucose uptake were evaluated. RESULTS: [18F]fluorodeoxyglucose uptake in the insula and amygdala of the intact hemisphere was significantly increased in response to the fearful context compared with the safe context. Such discrimination between the two contexts was not reflected in the activity of the insula-amygdala of the antOFC or vlPFC-lesioned hemisphere. Instead, uptake was at an intermediate level in both contexts. CONCLUSIONS: These findings demonstrate that the distinct control functions of the antOFC and vlPFC converge on the same downstream targets to promote emotion regulation, taking us closer to a mechanistic understanding of different forms of anxiety.

Novel Primate Model of Serotonin Transporter Genetic Polymorphisms Associated with Gene Expression, Anxiety and Sensitivity to Antidepressants.

Genetic polymorphisms in the repeat upstream region of the serotonin transporter gene (SLC6A4) are associated with individual differences in stress reactivity, vulnerability to affective disorders, and response to pharmacotherapy. However, the molecular, neurodevelopmental and psychopharmacological mechanisms underlying the link between SLC6A4 polymorphisms and the emotionally vulnerable phenotype are not fully understood. Thus, using the marmoset monkey Callithrix jacchus we characterize here a new neurobiological model to help to address these questions. We first sequenced the marmoset SLC6A4 promoter and identified a double nucleotide polymorphism (-2053AC/CT) and two single-nucleotide polymorphisms (-2022C/T and -1592G/C) within the repeat upstream region. We showed their association with gene expression using in vivo quantitative PCR and with affective behavior using a primate test of anxiety (human intruder test). The low-expressing haplotype (AC/C/G) was linked with high anxiety while the high-expressing one (CT/T/C) was associated with an active coping strategy in response to threat. Pharmacological challenge with an acute dose of the selective serotonin reuptake inhibitor, citalopram, revealed a genotype-dependent behavioral response. While individuals homozygous for the high anxiety-related haplotype AC/C/G exhibited a dose-dependent, anxiogenic response, individuals homozygous for the low anxiety-related haplotype CT/T/C showed an opposing, dose-dependent anxiolytic effect. These findings provide a novel genetic and behavioral primate model to study the molecular, neurodevelopmental, and psychopharmacological mechanisms that underlie genetic variation-associated complex behaviors, with specific implications for the understanding of normal and abnormal serotonin actions and the development of personalized pharmacological treatments for psychiatric disorders.

Serotonergic, brain volume and attentional correlates of trait anxiety in primates.

Trait anxiety is a risk factor for the development and maintenance of affective disorders, and insights into the underlying brain mechanisms are vital for improving treatment and prevention strategies. Translational studies in non-human primates, where targeted neurochemical and genetic manipulations can be made, are critical in view of their close neuroanatomical similarity to humans in brain regions implicated in trait anxiety. Thus, we characterised the serotonergic and regional brain volume correlates of trait-like anxiety in the marmoset monkey. Low- and high-anxious animals were identified by behavioral responses to a human intruder (HI) that are known to be sensitive to anxiolytic drug treatment. Extracellular serotonin levels within the amygdala were measured with in vivo microdialysis, at baseline and in response to challenge with the selective serotonin reuptake inhibitor, citalopram. Regional brain volume was assessed by structural magnetic resonance imaging. Anxious individuals showed persistent, long-term fearful responses to both a HI and a model snake, alongside sustained attention (vigilance) to novel cues in a context associated with unpredictable threat. Neurally, high-anxious marmosets showed reduced amygdala serotonin levels, and smaller volumes in a closely connected prefrontal region, the dorsal anterior cingulate cortex. These findings highlight behavioral and neural similarities between trait-like anxiety in marmosets and humans, and set the stage for further investigation of the processes contributing to vulnerability and resilience to affective disorders.

Individual differences in behavioral and cardiovascular reactivity to emotive stimuli and their relationship to cognitive flexibility in a primate model of trait anxiety.

High trait anxiety is a risk factor for the development of anxiety disorders. Like the disorders themselves high trait anxiety has marked phenotypic variation at the level of symptomatology and neural circuits, suggesting that there may be different symptoms and distinct neural circuits associated with risk for these disorders. To address these issues, it is essential to develop reliable animal models of trait anxiety in a non-human primate whose brain bears structural and functional similarity to humans. The present study investigated individual variation in responsivity to fearful and anxiety provoking stimuli in the common marmoset monkey. Seven out of 27 animals failed to display discriminative, conditioned cardiovascular and behavioral responses on an auditory fear discrimination task, similar to that seen in high anxious humans and rodents. Their heightened emotionality to a rubber snake was consistent with the hypothesis that they were high in trait-like anxiety. Evidence for phenotypic variation in the high anxiety group was provided by the finding that discrimination failure was predicted early in conditioning by either hyper-vigilant scanning to the cues or a reduction in blood pressure to the context, i.e., test apparatus. Given that high trait anxiety in humans can be associated with altered prefrontal cognitive functioning and previously we implicated the marmoset anterior orbitofrontal (antOFC) and ventrolateral prefrontal cortex (vlPFC) in negative emotion regulation, we also tested the marmosets on two tests of cognitive flexibility differentially dependent on these two regions. While the high anxious group did not differ overall in their perseverative performance, the two distinct phenotypes were differentially correlated with reduced perseverative responding on the OFC- and vlPFC-dependent flexibility tests. Together, this study provides a new model of trait anxiety in marmosets amenable to analysis of phenotypic variation and neural circuitry.