DREADD-mediated amygdala activation is sufficient to induce anxiety-like responses in young nonhuman primates.

Anxiety disorders are among the most prevalent psychiatric disorders, with symptoms often beginning early in life. To model the pathophysiology of human pathological anxiety, we utilized Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) in a nonhuman primate model of anxious temperament to selectively increase neuronal activity of the amygdala. Subjects included 10 young rhesus macaques; 5 received bilateral infusions of AAV5-hSyn-HA-hM3Dq into the dorsal amygdala, and 5 served as controls. Subjects underwent behavioral testing in the human intruder paradigm following clozapine or vehicle administration, prior to and following surgery. Behavioral results indicated that clozapine treatment post-surgery increased freezing across different threat-related contexts in hM3Dq subjects. This effect was again observed approximately 1.9 years following surgery, indicating the long-term functional capacity of DREADD-induced neuronal activation. [11C]deschloroclozapine PET imaging demonstrated amygdala hM3Dq-HA specific binding, and immunohistochemistry revealed that hM3Dq-HA expression was most prominent in basolateral nuclei. Electron microscopy confirmed expression was predominantly on neuronal membranes. Together, these data demonstrate that activation of primate amygdala neurons is sufficient to induce increased anxiety-related behaviors, which could serve as a model to investigate pathological anxiety in humans.

DREADD-mediated amygdala activation is sufficient to induce anxiety-like responses in young nonhuman primates.

Anxiety disorders are among the most prevalent psychiatric disorders, with symptoms often beginning early in life. To model the pathophysiology of human pathological anxiety, we utilized Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) in a nonhuman primate model of anxious temperament to selectively increase neuronal activity of the amygdala. Subjects included 10 young rhesus macaques; 5 received bilateral infusions of AAV5-hSyn-HA-hM3Dq into the dorsal amygdala, and 5 served as controls. Subjects underwent behavioral testing in the human intruder paradigm following clozapine or vehicle administration, prior to and following surgery. Behavioral results indicated that clozapine treatment post-surgery increased freezing across different threat-related contexts in hM3Dq subjects. This effect was again observed approximately 1.9 years following surgery, indicating the long-term functional capacity of DREADD-induced neuronal activation. [11C]deschloroclozapine PET imaging demonstrated amygdala hM3Dq-HA specific binding, and immunohistochemistry revealed that hM3Dq-HA expression was most prominent in basolateral nuclei. Electron microscopy confirmed expression was predominantly on neuronal membranes. Together, these data demonstrate that activation of primate amygdala neurons is sufficient to induce increased anxiety-related behaviors, which could serve as a model to investigate pathological anxiety in humans.

Real-time trajectory guide tracking for intraoperative MRI-guided neurosurgery.

PURPOSE: In current intraoperative MRI (IMRI) methods, an iterative approach is used to aim trajectory guides at intracerebral targets: image MR-visible features, determine current aim by fitting model to image, manipulate device, repeat. Infrequent updates are produced by such methods, compared to rapid optically tracked stereotaxy used in the operating room. Our goal was to develop a real-time interactive IMRI method for aiming. METHODS: The current trajectory was computed from two points along the guide's central axis, rather than by imaging the entire device. These points were determined by correlating one-dimensional spokes from a radial sequence with the known cross-sectional projection of the guide. The real-time platform RTHawk was utilized to control MR sequences and data acquisition. On-screen updates were viewed by the operator while simultaneously manipulating the guide to align it with the planned trajectory. Accuracy was quantitated in a phantom, and in vivo validation was demonstrated in nonhuman primates undergoing preclinical gene ( n = 5 $$ n=5 $$ ) and cell ( n = 4 $$ n=4 $$ ) delivery surgeries. RESULTS: Updates were produced at 5 Hz In 10 phantom experiments at a depth of 48 mm, the cannula tip was placed with radial error of (min, mean, max) = (0.16, 0.29, 0.68) mm. Successful in vivo delivery of payloads to all 14 targets was demonstrated across nine surgeries with depths of (min, mean, max) = (33.3, 37.9, 42.5) mm. CONCLUSION: A real-time interactive update rate was achieved, reducing operator fatigue without compromising accuracy. Qualitative interpretation of images during aiming was rendered unnecessary by objectively computing device alignment.

Evidence in primates supporting the use of chemogenetics for the treatment of human refractory neuropsychiatric disorders.

Non-human primate (NHP) models are essential for developing and translating new treatments that target neural circuit dysfunction underlying human psychopathology. As a proof-of-concept for treating neuropsychiatric disorders, we used a NHP model of pathological anxiety to investigate the feasibility of decreasing anxiety by chemogenetically (DREADDs [designer receptors exclusively activated by designer drugs]) reducing amygdala neuronal activity. Intraoperative MRI surgery was used to infect dorsal amygdala neurons with AAV5-hSyn-HA-hM4Di in young rhesus monkeys. In vivo microPET studies with [11C]-deschloroclozapine and postmortem autoradiography with [3H]-clozapine demonstrated selective hM4Di binding in the amygdala, and neuronal expression of hM4Di was confirmed with immunohistochemistry. Additionally, because of its high affinity for DREADDs, and its approved use in humans, we developed an individualized, low-dose clozapine administration strategy to induce DREADD-mediated amygdala inhibition. Compared to controls, clozapine selectively decreased anxiety-related freezing behavior in the human intruder paradigm in hM4Di-expressing monkeys, while coo vocalizations and locomotion were unaffected. These results are an important step in establishing chemogenetic strategies for patients with refractory neuropsychiatric disorders in which amygdala alterations are central to disease pathophysiology.

[18F]FEPPA PET imaging for monitoring CD68-positive microglia/macrophage neuroinflammation in nonhuman primates.

PURPOSE: The aim of this study was to examine whether the translocator protein 18-kDa (TSPO) PET ligand [18F]FEPPA has the sensitivity for detecting changes in CD68-positive microglial/macrophage activation in hemiparkinsonian rhesus macaques treated with allogeneic grafts of induced pluripotent stem cell-derived midbrain dopaminergic neurons (iPSC-mDA). METHODS: In vivo positron emission tomography (PET) imaging with [18F]FEPPA was used in conjunction with postmortem CD68 immunostaining to evaluate neuroinflammation in the brains of hemiparkinsonian rhesus macaques (n = 6) that received allogeneic iPSC-mDA grafts in the putamen ipsilateral to MPTP administration. RESULTS: Based on assessment of radiotracer uptake and confirmed by visual inspection of the imaging data, nonhuman primates with allogeneic grafts showed increased [18F]FEPPA binding at the graft sites relative to the contralateral putamen. From PET asymmetry analysis of the images, the mean asymmetry index of the monkeys was AI = - 0.085 ± 0.018. Evaluation and scoring of CD68 immunoreactivity by an investigator blind to the treatment identified significantly more neuroinflammation in the grafted areas of the putamen compared to the contralateral putamen (p = 0.0004). [18F]FEPPA PET AI showed a positive correlation with CD68 immunoreactivity AI ratings in the monkeys (Spearman's ρ = 0.94; p = 0.005). CONCLUSION: These findings reveal that [18F]FEPPA PET is an effective marker for detecting increased CD68-positive microglial/macrophage activation and demonstrates sufficient sensitivity to detect changes in neuroinflammation in vivo following allogeneic cell engraftment.

Dorsal Amygdala Neurotrophin-3 Decreases Anxious Temperament in Primates.

BACKGROUND: An early-life anxious temperament (AT) is a risk factor for the development of anxiety, depression, and comorbid substance abuse. We validated a nonhuman primate model of early-life AT and identified the dorsal amygdala as a core component of AT's neural circuit. Here, we combine RNA sequencing, viral-vector gene manipulation, functional brain imaging, and behavioral phenotyping to uncover AT's molecular substrates. METHODS: In response to potential threat, AT and brain metabolism were assessed in 46 young rhesus monkeys. We identified AT-related transcripts using RNA-sequencing data from dorsal amygdala tissue (including central nucleus of the amygdala [Ce] and dorsal regions of the basal nucleus). Based on the results, we overexpressed the neurotrophin-3 gene, NTF3, in the dorsal amygdala using intraoperative magnetic resonance imaging-guided surgery (n = 5 per group). RESULTS: This discovery-based approach identified AT-related alterations in the expression of well-established and novel genes, including an inverse association between NTRK3 expression and AT. NTRK3 is an interesting target because it is a relatively unexplored neurotrophic factor that modulates intracellular neuroplasticity pathways. Overexpression of the transcript for NTRK3's endogenous ligand, NTF3, in the dorsal amygdala resulted in reduced AT and altered function in AT's neural circuit. CONCLUSIONS: Together, these data implicate neurotrophin-3/NTRK3 signaling in the dorsal amygdala in mediating primate anxiety. More generally, this approach provides an important step toward understanding the molecular underpinnings of early-life AT and will be useful in guiding the development of treatments to prevent the development of stress-related psychopathology.

Overexpressing Corticotropin-Releasing Factor in the Primate Amygdala Increases Anxious Temperament and Alters Its Neural Circuit.

BACKGROUND: Nonhuman primate models are critical for understanding mechanisms underlying human psychopathology. We established a nonhuman primate model of anxious temperament (AT) for studying the early-life risk to develop anxiety and depression. Studies have identified the central nucleus of the amygdala (Ce) as an essential component of AT's neural substrates. Corticotropin-releasing factor (CRF) is expressed in the Ce, has a role in stress, and is linked to psychopathology. Here, in young rhesus monkeys, we combined viral vector technology with assessments of anxiety and multimodal neuroimaging to understand the consequences of chronically increased CRF in the Ce region. METHODS: Using real-time intraoperative magnetic resonance imaging-guided convection-enhanced delivery, five monkeys received bilateral dorsal amygdala Ce-region infusions of adeno-associated virus serotype 2 containing the CRF construct. Their cagemates served as unoperated control subjects. AT, regional brain metabolism, resting functional magnetic resonance imaging, and diffusion tensor imaging were assessed before and 2 months after viral infusions. RESULTS: Dorsal amygdala CRF overexpression significantly increased AT and metabolism within the dorsal amygdala. Additionally, we observed changes in metabolism in other AT-related regions, as well as in measures of functional and structural connectivity. CONCLUSIONS: This study provides a translational roadmap that is important for understanding human psychopathology by combining molecular manipulations used in rodents with behavioral phenotyping and multimodal neuroimaging measures used in humans. The results indicate that chronic CRF overexpression in primates not only increases AT but also affects metabolism and connectivity within components of AT's neural circuitry.