Spatiotemporal expression patterns of anxiety disorder-associated genes.

Anxiety disorders (ADs) are the most common form of mental disorder that affects millions of individuals worldwide. Although physiological studies have revealed the neural circuits related to AD symptoms, how AD-associated genes are spatiotemporally expressed in the human brain still remains unclear. In this study, we integrated genome-wide association studies of four human AD subtypes-generalized anxiety disorder, social anxiety disorder, panic disorder, and obsessive-compulsive disorder-with spatial gene expression patterns. Our investigation uncovered a novel division among AD-associated genes, marked by significant and distinct expression enrichments in the cerebral nuclei, limbic, and midbrain regions. Each gene cluster was associated with specific anxiety-related behaviors, signaling pathways, region-specific gene networks, and cell types. Notably, we observed a significant negative correlation in the temporal expression patterns of these gene clusters during various developmental stages. Moreover, the specific brain regions enriched in each gene group aligned with neural circuits previously associated with negative decision-making and anxious temperament. These results suggest that the two distinct gene clusters may underlie separate neural systems involved in anxiety. As a result, our findings bridge the gap between genes and neural circuitry, shedding light on the mechanisms underlying AD-associated behaviors.

Multiplexed action-outcome representation by striatal striosome-matrix compartments detected with a mouse cost-benefit foraging task.

Learning about positive and negative outcomes of actions is crucial for survival and underpinned by conserved circuits including the striatum. How associations between actions and outcomes are formed is not fully understood, particularly when the outcomes have mixed positive and negative features. We developed a novel foraging ('bandit') task requiring mice to maximize rewards while minimizing punishments. By 2-photon Ca++ imaging, we monitored activity of visually identified anterodorsal striatal striosomal and matrix neurons. We found that action-outcome associations for reward and punishment were encoded in parallel in partially overlapping populations. Single neurons could, for one action, encode outcomes of opposing valence. Striosome compartments consistently exhibited stronger representations of reinforcement outcomes than matrix, especially for high reward or punishment prediction errors. These findings demonstrate multiplexing of action-outcome contingencies by single identified striatal neurons and suggest that striosomal neurons are particularly important in action-outcome learning.

The Role of the Dorsal-Lateral Prefrontal Cortex in Reward Sensitivity During Approach-Avoidance Conflict.

Approach-Avoidance conflict (AAC) arises from decisions with embedded positive and negative outcomes, such that approaching leads to reward and punishment and avoiding to neither. Despite its importance, the field lacks a mechanistic understanding of which regions are driving avoidance behavior during conflict. In the current task, we utilized transcranial magnetic stimulation (TMS) and drift-diffusion modeling to investigate the role of one of the most prominent regions relevant to AAC-the dorsolateral prefrontal cortex (dlPFC). The first experiment uses in-task disruption to examine the right dlPFC's (r-dlPFC) causal role in avoidance behavior. The second uses single TMS pulses to probe the excitability of the r-dlPFC, and downstream cortical activations, during avoidance behavior. Disrupting r-dlPFC during conflict decision-making reduced reward sensitivity. Further, r-dlPFC was engaged with a network of regions within the lateral and medial prefrontal, cingulate, and temporal cortices that associate with behavior during conflict. Together, these studies use TMS to demonstrate a role for the dlPFC in reward sensitivity during conflict and elucidate the r-dlPFC's network of cortical regions associated with avoidance behavior. By identifying r-dlPFC's mechanistic role in AAC behavior, contextualized within its conflict-specific downstream neural connectivity, we advance dlPFC as a potential neural target for psychiatric therapeutics.

Generalized and social anxiety disorder interactomes show distinctive overlaps with striosome and matrix interactomes.

Mechanisms underlying anxiety disorders remain elusive despite the discovery of several associated genes. We constructed the protein-protein interaction networks (interactomes) of six anxiety disorders and noted enrichment for striatal expression among common genes in the interactomes. Five of these interactomes shared distinctive overlaps with the interactomes of genes that were differentially expressed in two striatal compartments (striosomes and matrix). Generalized anxiety disorder and social anxiety disorder interactomes showed exclusive and statistically significant overlaps with the striosome and matrix interactomes, respectively. Systematic gene expression analysis with the anxiety disorder interactomes constrained to contain only those genes that were shared with striatal compartment interactomes revealed a bifurcation among the disorders, which was influenced by the anterior cingulate cortex, nucleus accumbens, amygdala and hippocampus, and the dopaminergic signaling pathway. Our results indicate that the functionally distinct striatal pathways constituted by the striosome and the matrix may influence the etiological differentiation of various anxiety disorders.

Causal Evidence for Induction of Pessimistic Decision-Making in Primates by the Network of Frontal Cortex and Striosomes.

Clinical studies have shown that patients with anxiety disorders exhibited coactivation of limbic cortices and basal ganglia, which together form a large-scale brain network. The mechanisms by which such a large-scale network could induce or modulate anxiety-like states are largely unknown. This article reviews our experimental program in macaques demonstrating a causal involvement of local striatal and frontal cortical sites in inducing pessimistic decision-making that underlies anxiety. Where relevant, we related these findings to the wider literature. To identify such sites, we have made a series of methodologic advances, including the combination of causal evidence for behavioral modification of pessimistic decisions with viral tracing methods. Critically, we introduced a version of the classic approach-avoidance (Ap-Av) conflict task, modified for use in non-human primates. We performed microstimulation of limbic-related cortical regions and the striatum, focusing on the pregenual anterior cingulate cortex (pACC), the caudal orbitofrontal cortex (cOFC), and the caudate nucleus (CN). Microstimulation of localized sites within these regions induced pessimistic decision-making by the monkeys, supporting the idea that the focal activation of these regions could induce an anxiety-like state, which subsequently influences decision-making. We further performed combined microstimulation and tract-tracing experiments by injecting anterograde viral tracers into focal regions, at which microstimulation induced increased avoidance. We found that effective stimulation sites in both pACC and cOFC zones projected preferentially to striosomes in the anterior striatum. Experiments in rodents have shown that the striosomes in the anterior striatum project directly to the dopamine-containing cells in the substantia nigra, and we have found evidence for a functional connection between striosomes and the lateral habenular region in which responses to reward are inhibitory. We present here further evidence for network interactions: we show that the pACC and cOFC project to common structures, including not only the anterior parts of the striosome compartment but also the tail of the CN, the subgenual ACC, the amygdala, and the thalamus. Together, our findings suggest that networks having pACC and cOFC as nodes share similar features in their connectivity patterns. We here hypothesize, based on these results, that the brain sites related to pessimistic judgment are mediated by a large-scale brain network that regulates dopaminergic functions and includes striosomes and striosome-projecting cortical regions.

Computational phenotyping of brain-behavior dynamics underlying approach-avoidance conflict in major depressive disorder.

Adaptive behavior requires balancing approach and avoidance based on the rewarding and aversive consequences of actions. Imbalances in this evaluation are thought to characterize mood disorders such as major depressive disorder (MDD). We present a novel application of the drift diffusion model (DDM) suited to quantify how offers of reward and aversiveness, and neural correlates thereof, are dynamically integrated to form decisions, and how such processes are altered in MDD. Hierarchical parameter estimation from the DDM demonstrated that the MDD group differed in three distinct reward-related parameters driving approach-based decision making. First, MDD was associated with reduced reward sensitivity, measured as the impact of offered reward on evidence accumulation. Notably, this effect was replicated in a follow-up study. Second, the MDD group showed lower starting point bias towards approaching offers. Third, this starting point was influenced in opposite directions by Pavlovian effects and by nucleus accumbens activity across the groups: greater accumbens activity was related to approach bias in controls but avoid bias in MDD. Cross-validation revealed that the combination of these computational biomarkers were diagnostic of patient status, with accumbens influences being particularly diagnostic. Finally, within the MDD group, reward sensitivity and nucleus accumbens parameters were differentially related to symptoms of perceived stress and depression. Collectively, these findings establish the promise of computational psychiatry approaches to dissecting approach-avoidance decision dynamics relevant for affective disorders.

Striatal Beta Oscillation and Neuronal Activity in the Primate Caudate Nucleus Differentially Represent Valence and Arousal Under Approach-Avoidance Conflict.

An approach-avoidance (Ap-Av) conflict arises when an individual has to decide whether to accept or reject a compound offer that has features indicating both reward and punishment. During value judgments of likes and dislikes, arousal responses simultaneously emerge and influence reaction times and the frequency of behavioral errors. In Ap-Av decision-making, reward and punishment differentially influence valence and arousal, allowing us to dissociate their neural processing. The primate caudate nucleus (CN) has been implicated in affective judgment, but it is still unclear how neural responses in the CN represent decision-related variables underlying choice. To address this issue, we recorded spikes and local field potentials (LFPs) from the CN while macaque monkeys performed an Ap-Av decision-making task. We analyzed 450 neuronal units and 667 beta oscillatory activities recorded during the performance of the task. To examine how these activities represented valence, we focused on beta-band responses and unit activities that encoded the chosen value (ChV) of the compound offer as derived from an econometric model. Unit activities exhibited either positive (65.0% = 26/40) or negative (35.0% = 14/40) correlations with the ChV, whereas beta responses exhibited almost exclusively positive correlations with the ChV (98.4% = 62/63). We examined arousal representation by focusing on beta responses and unit activities that encoded the frequency of omission errors (FOE), which were negatively correlated with arousal. The unit activities were either positively (65.3% = 17/26) or negatively (34.6% = 9/26) correlated with the FOE, whereas the beta responses were almost entirely positively correlated with the FOE (95.8% = 23/24). We found that the temporal onset of the beta-band responses occurred sequentially across conditions: first, the negative-value, then low-arousal, and finally, high-value conditions. These findings suggest the distinctive roles of CN beta oscillations that were sequentially activated for the valence and arousal conditions. By identifying dissociable groups of CN beta-band activity responding in relation to valence and arousal, we demonstrate that the beta responses mainly exhibited selective activation for the high-valence and low-arousal conditions, whereas the unit activities simultaneously recorded in the same experiments responded to chosen value and other features of decision-making under approach-avoidance conflict.

Microstimulation of primate neocortex targeting striosomes induces negative decision-making.

Here, we combined MRI-guided electrical microstimulation and viral tracing to examine the function of a corticostriatal circuit implicated by previous cortical microstimulation as modulating affective judgment and decision-making. Local microstimulation of a small part of the pregenual anterior cingulate cortex (pACC) was found to increase avoidance decisions in a cost-benefit decision-making task (Ap-Av task) in which differing amounts of "good" and "bad" options were given simultaneously. No effect of such stimulation was found when the monkeys performed a task in which both offers were rewarding, but given in different amounts. We asked whether we could identify the targets of such corticostriatal circuits when the cortical microstimulation sites were explicitly identified as affecting approach or avoidance in the Ap-Av task. We explored the pACC and caudal orbitofrontal cortex (cOFC) to look for such sites. For each cortical region, we found sites at which microstimulation induced increased avoidance behavior. After identifying these sites, we injected viral tracers carrying constructs allowing subsequent track-tracing post-mortem. For each site identified behaviorally as increasing avoidance choices, we found strong fiber projections to the anterior striatum with large parts of these targeting striosomes subsequently identified by serial section immunohistochemistry. With fMRI, we demonstrated that microstimulation in an anesthetized monkey at sites pre-identified as affecting Ap-Av choices induced blood oxygen level dependent activation of the anterior striatum, confirming that the microstimulation method that we applied was effective in activating the striatum. These findings outline circuits leading from pACC/cOFC to striosomes and causally modulating decision-making under emotional conflict.

Approach-Avoidance Conflict in Major Depressive Disorder: Congruent Neural Findings in Humans and Nonhuman Primates.

BACKGROUND: Maladaptive approach-avoidance behavior has been implicated in the pathophysiology of major depressive disorder (MDD), but the neural basis of these abnormalities in decision making remains unclear. Capitalizing on recent preclinical findings, we adapted an approach-avoidance conflict task from nonhuman primate research for use in human functional magnetic resonance imaging (fMRI). METHODS: Forty-two female participants, including 18 unmedicated individuals with current MDD (mean age 25.2 ± 5.1 years) and 24 psychiatrically healthy control subjects (mean age 26.3 ± 7.6 years) completed the adapted approach-avoidance task during fMRI. To probe potential mechanistic factors underlying the observed behavioral and fMRI findings and to inform interpretation of putative group differences, we examined electrophysiological data from 2 female Macaca mulatta monkeys performing the approach-avoidance conflict task mimicked in the fMRI study. RESULTS: Findings demonstrated congruent neural correlates of approach-avoidance conflict and aversive responsiveness in the anterior cingulate cortex, including the pregenual cortex, of human subjects and macaques (humans: p < .05 whole-brain corrected; macaques: p < .05). The MDD group exhibited aberrant task-related activations in the anterior cingulate cortex, prefrontal cortex, and striatum (all ps < .05). Neural effects in the MDD group were cross-sectionally associated with stress and depressive symptoms. Importantly, they also prospectively predicted stress at 6-month follow-up (all ps < .05). CONCLUSIONS: Findings indicate that there is conservation of anterior cingulate activation across species and that frontal and striatal regions, in unmedicated humans with MDD, are abnormally responsive during cost-benefit decision making. We suggest that these disruptions could be valuable candidates for translational biomarkers.

Predominant Striatal Input to the Lateral Habenula in Macaques Comes from Striosomes.

Striosomes, neurochemically specialized modules in the striatum, are thought to be nodes in circuits extending, via basal ganglia pathways, from mood-related neocortical regions to dopamine-containing neurons of the substantia nigra. Yet striosomes have remained beyond the reach of electrophysiological methods to identify them, especially in non-human primates. Such work is needed for translational as well as for basic science. Here we introduce a method to identify striosomes on-line in awake, behaving macaques. We combined electrical microstimulation of the striatum with simultaneous electrophysiological recording in the lateral habenula (LHb) followed by immunohistochemistry. We demonstrate that striosomes provide the predominant striatal input to the macaque pallido-habenular circuit, which is known to function in relation to reinforcement signaling. Further, our experiments suggest that striosomes from different striatal regions may convergently influence the lateral habenula. This work now opens the way to defining the functions of striosomes in behaving primates in relation to mood, motivation, and action.

Striatal Microstimulation Induces Persistent and Repetitive Negative Decision-Making Predicted by Striatal Beta-Band Oscillation.

Persistent thoughts inducing irrationally pessimistic and repetitive decisions are often symptoms of mood and anxiety disorders. Regional neural hyperactivities have been associated with these disorders, but it remains unclear whether there is a specific brain region causally involved in these persistent valuations. Here, we identified potential sources of such persistent states by microstimulating the striatum of macaques performing a task by which we could quantitatively estimate their subjective pessimistic states using their choices to accept or reject conflicting offers. We found that this microstimulation induced irrationally repetitive choices with negative evaluations. Local field potentials recorded in the same microstimulation sessions exhibited modulations of beta-band oscillatory activity that paralleled the persistent negative states influencing repetitive decisions. These findings demonstrate that local striatal zones can causally affect subjective states influencing persistent negative valuation and that abnormal beta-band oscillations can be associated with persistency in valuation accompanied by an anxiety-like state.

Miniaturized neural system for chronic, local intracerebral drug delivery.

Recent advances in medications for neurodegenerative disorders are expanding opportunities for improving the debilitating symptoms suffered by patients. Existing pharmacologic treatments, however, often rely on systemic drug administration, which result in broad drug distribution and consequent increased risk for toxicity. Given that many key neural circuitries have sub-cubic millimeter volumes and cell-specific characteristics, small-volume drug administration into affected brain areas with minimal diffusion and leakage is essential. We report the development of an implantable, remotely controllable, miniaturized neural drug delivery system permitting dynamic adjustment of therapy with pinpoint spatial accuracy. We demonstrate that this device can chemically modulate local neuronal activity in small (rodent) and large (nonhuman primate) animal models, while simultaneously allowing the recording of neural activity to enable feedback control.

Chronic Stress Alters Striosome-Circuit Dynamics, Leading to Aberrant Decision-Making.

Effective evaluation of costs and benefits is a core survival capacity that in humans is considered as optimal, "rational" decision-making. This capacity is vulnerable in neuropsychiatric disorders and in the aftermath of chronic stress, in which aberrant choices and high-risk behaviors occur. We report that chronic stress exposure in rodents produces abnormal evaluation of costs and benefits resembling non-optimal decision-making in which choices of high-cost/high-reward options are sharply increased. Concomitantly, alterations in the task-related spike activity of medial prefrontal neurons correspond with increased activity of their striosome-predominant striatal projection neuron targets and with decreased and delayed striatal fast-firing interneuron activity. These effects of chronic stress on prefronto-striatal circuit dynamics could be blocked or be mimicked by selective optogenetic manipulation of these circuits. We suggest that altered excitation-inhibition dynamics of striosome-based circuit function could be an underlying mechanism by which chronic stress contributes to disorders characterized by aberrant decision-making under conflict. VIDEO ABSTRACT.

Long-term dopamine neurochemical monitoring in primates.

Many debilitating neuropsychiatric and neurodegenerative disorders are characterized by dopamine neurotransmitter dysregulation. Monitoring subsecond dopamine release accurately and for extended, clinically relevant timescales is a critical unmet need. Especially valuable has been the development of electrochemical fast-scan cyclic voltammetry implementing microsized carbon fiber probe implants to record fast millisecond changes in dopamine concentrations. Nevertheless, these well-established methods have only been applied in primates with acutely (few hours) implanted sensors. Neurochemical monitoring for long timescales is necessary to improve diagnostic and therapeutic procedures for a wide range of neurological disorders. Strategies for the chronic use of such sensors have recently been established successfully in rodents, but new infrastructures are needed to enable these strategies in primates. Here we report an integrated neurochemical recording platform for monitoring dopamine release from sensors chronically implanted in deep brain structures of nonhuman primates for over 100 days, together with results for behavior-related and stimulation-induced dopamine release. From these chronically implanted probes, we measured dopamine release from multiple sites in the striatum as induced by behavioral performance and reward-related stimuli, by direct stimulation, and by drug administration. We further developed algorithms to automate detection of dopamine. These algorithms could be used to track the effects of drugs on endogenous dopamine neurotransmission, as well as to evaluate the long-term performance of the chronically implanted sensors. Our chronic measurements demonstrate the feasibility of measuring subsecond dopamine release from deep brain circuits of awake, behaving primates in a longitudinally reproducible manner.

Habit Learning by Naive Macaques Is Marked by Response Sharpening of Striatal Neurons Representing the Cost and Outcome of Acquired Action Sequences.

Over a century of scientific work has focused on defining the factors motivating behavioral learning. Observations in animals and humans trained on a wide range of tasks support reinforcement learning (RL) algorithms as accounting for the learning. Still unknown, however, are the signals that drive learning in naive, untrained subjects. Here, we capitalized on a sequential saccade task in which macaque monkeys acquired repetitive scanning sequences without instruction. We found that spike activity in the caudate nucleus after each trial corresponded to an integrated cost-benefit signal that was highly correlated with the degree of naturalistic untutored learning by the monkeys. Across learning, neurons encoding both cost and outcome gradually acquired increasingly sharp phasic trial-end responses that paralleled the development of the habit-like, repetitive saccade sequences. Our findings demonstrate an integrated cost-benefit signal by which RL and its neural correlates could drive naturalistic behaviors in freely behaving primates.

A Corticostriatal Path Targeting Striosomes Controls Decision-Making under Conflict.

A striking neurochemical form of compartmentalization has been found in the striatum of humans and other species, dividing it into striosomes and matrix. The function of this organization has been unclear, but the anatomical connections of striosomes indicate their relation to emotion-related brain regions, including the medial prefrontal cortex. We capitalized on this fact by combining pathway-specific optogenetics and electrophysiology in behaving rats to search for selective functions of striosomes. We demonstrate that a medial prefronto-striosomal circuit is selectively active in and causally necessary for cost-benefit decision-making under approach-avoidance conflict conditions known to evoke anxiety in humans. We show that this circuit has unique dynamic properties likely reflecting striatal interneuron function. These findings demonstrate that cognitive and emotion-related functions are, like sensory-motor processing, subject to encoding within compartmentally organized representations in the forebrain and suggest that striosome-targeting corticostriatal circuits can underlie neural processing of decisions fundamental for survival.

Motivation and affective judgments differentially recruit neurons in the primate dorsolateral prefrontal and anterior cingulate cortex.

The judgment of whether to accept or to reject an offer is determined by positive and negative affect related to the offer, but affect also induces motivational responses. Rewarding and aversive cues influence the firing rates of many neurons in primate prefrontal and cingulate neocortical regions, but it still is unclear whether neurons in these regions are related to affective judgment or to motivation. To address this issue, we recorded simultaneously the neuronal spike activities of single units in the dorsolateral prefrontal cortex (dlPFC) and the anterior cingulate cortex (ACC) of macaque monkeys as they performed approach-avoidance (Ap-Av) and approach-approach (Ap-Ap) decision-making tasks that can behaviorally dissociate affective judgment and motivation. Notably, neurons having activity correlated with motivational condition could be distinguished from neurons having activity related to affective judgment, especially in the Ap-Av task. Although many neurons in both regions exhibited similar, selective patterns of task-related activity, we found a larger proportion of neurons activated in low motivational conditions in the dlPFC than in the ACC, and the onset of this activity was significantly earlier in the dlPFC than in the ACC. Furthermore, the temporal onsets of affective judgment represented by neuronal activities were significantly slower in the low motivational conditions than in the other conditions. These findings suggest that motivation and affective judgment both recruit dlPFC and ACC neurons but with differential degrees of involvement and timing.

A non-invasive head-holding device for chronic neural recordings in awake behaving monkeys.

BACKGROUND: We have developed a novel head-holding device for behaving non-human primates that affords stability suitable for reliable chronic electrophysiological recording experiments. The device is completely non-invasive, and thus avoids the risk of infection and other complications that can occur with the use of conventional, surgically implanted head-fixation devices. NEW METHOD: The device consists of a novel non-invasive head mold and bar clamp holder, and is customized to the shape of each monkey's head. The head-holding device that we introduce, combined with our recording system and reflection-based eye-tracking system, allows for chronic behavioral experiments and single-electrode or multi-electrode recording, as well as manipulation of brain activity. RESULTS AND COMPARISON WITH EXISTING METHODS: With electrodes implanted chronically in multiple brain regions, we could record neural activity from cortical and subcortical structures with stability equal to that recorded with conventional head-post fixation. Consistent with the non-invasive nature of the device, we could record neural signals for more than two years with a single implant. Importantly, the monkeys were able to hold stable eye fixation positions while held by this device, demonstrating the possibility of analyzing eye movement data with only the gentle restraint imposed by the non-invasive head-holding device. CONCLUSIONS: We show that the head-holding device introduced here can be extended to the head holding of smaller animals, and note that it could readily be adapted for magnetic resonance brain imaging over extended periods of time.

Localized microstimulation of primate pregenual cingulate cortex induces negative decision-making.

The pregenual anterior cingulate cortex (pACC) has been implicated in human anxiety disorders and depression, but the circuit-level mechanisms underlying these disorders are unclear. In healthy individuals, the pACC is involved in cost-benefit evaluation. We developed a macaque version of an approach-avoidance decision task used to evaluate anxiety and depression in humans and, with multi-electrode recording and cortical microstimulation, we probed pACC function as monkeys performed this task. We found that the macaque pACC has an opponent process-like organization of neurons representing motivationally positive and negative subjective value. Spatial distribution of these two neuronal populations overlapped in the pACC, except in one subzone, where neurons with negative coding were more numerous. Notably, microstimulation in this subzone, but not elsewhere in the pACC, increased negative decision-making, and this negative biasing was blocked by anti-anxiety drug treatment. This cortical zone could be critical for regulating negative emotional valence and anxiety in decision-making.

A system for recording neural activity chronically and simultaneously from multiple cortical and subcortical regions in nonhuman primates.

A major goal of neuroscience is to understand the functions of networks of neurons in cognition and behavior. Recent work has focused on implanting arrays of ∼100 immovable electrodes or smaller numbers of individually adjustable electrodes, designed to target a few cortical areas. We have developed a recording system that allows the independent movement of hundreds of electrodes chronically implanted in several cortical and subcortical structures. We have tested this system in macaque monkeys, recording simultaneously from up to 127 electrodes in 14 brain regions for up to one year at a time. A key advantage of the system is that it can be used to sample different combinations of sites over prolonged periods, generating multiple snapshots of network activity from a single implant. Used in conjunction with microstimulation and injection methods, this versatile system represents a powerful tool for studying neural network activity in the primate brain.