Evaluation of [18F]fluoroestradiol and ChRERα as a gene expression PET reporter system in rhesus monkey brain.

Positron emission tomography (PET) reporter systems are a valuable means of estimating the level of expression of a transgene in vivo. For example, the safety and efficacy of gene therapy approaches for the treatment of neurological and neuropsychiatric disorders could be enhanced via the monitoring of exogenous gene expression levels in the brain. The present study evaluated the ability of a newly developed PET reporter system [18F]fluoroestradiol ([18F]FES) and the estrogen receptor-based PET reporter ChRERα, to monitor expression levels of a small hairpin RNA (shRNA) designed to suppress choline acetyltransferase (ChAT) expression in rhesus monkey brain. The ChRERα gene and shRNA were expressed from the same transcript via lentivirus injected into monkey striatum. In two monkeys that received injections of viral vector, [18F]FES binding increased by 70% and 86% at the target sites compared with pre-injection, demonstrating that ChRERα expression could be visualized in vivo with PET imaging. Post-mortem immunohistochemistry confirmed that ChAT expression was significantly suppressed in regions in which [18F]FES uptake was increased. The consistency between PET imaging and immunohistochemical results suggests that [18F]FES and ChRERα can serve as a PET reporter system in rhesus monkey brain for in vivo evaluation of the expression of potential therapeutic agents, such as shRNAs.

Visual recognition in rhesus monkeys requires area TE but not TEO.

The primate visual system is often described as a hierarchical feature-conjunction pathway, whereby each level represents an increasingly complex combination of image elements, culminating in the representation of whole coherent images in anterior inferior temporal cortex. Although many models of the ventral visual stream emphasize serial feedforward processing ((Poggio T, Mutch J, Leibo J, Rosasco L, Tacchetti A. The computationalmagic of the ventral stream: sketch of a theory (and why some deep architectures work). TechRep MIT-CSAIL-TR-2012-035. MIT CSAIL, Cambridge, MA. 2012); (Yamins DLK, DiCarlo JJ. Eight open questions in the computational modeling of higher sensory cortex. Curr Opin Neurobiol. 2016:37:114-120.)), anatomical studies show connections that bypass intermediate areas and that feedback to preceding areas ((Distler C, Boussaoud D, Desimone R, Ungerleider LG. Cortical connections of inferior temporal area TEO in macaque monkeys. J Comp Neurol. 1993:334(1):125-150.); (Kravitz DJ, Saleem KS, Baker CI, Mishkin M. A new neural framework for visuospatial processing. Nat Rev Neurosci. 2011:12(4):217-230.)). Prior studies on visual discrimination and object transforms also provide evidence against a strictly feed-forward serial transfer of information between adjacent areas ((Kikuchi R, Iwai E. The locus of the posterior subdivision of the inferotemporal visual learning area in the monkey. Brain Res. 1980:198(2):347-360.); (Weiskrantz L, Saunders RC. Impairments of visual object transforms in monkeys. Brain. 1984:107(4):1033-1072.); (Kar K, DiCarlo JJ. Fast recurrent processing via ventrolateral prefrontal cortex is needed by the primate ventral stream for robust Core visual object recognition. Neuron. 2021:109(1):164-176.e5.)). Thus, we sought to investigate whether behaviorally relevant propagation of visual information is as strictly sequential as sometimes supposed. We compared the accuracy of visual recognition after selective removal of specific subregions of inferior temporal cortex-area TEO, area TE, or both areas combined. Removal of TEO alone had no detectable effect on recognition memory, whereas removal of TE alone produced a large and significant impairment. Combined removal of both areas created no additional deficit relative to removal of TE alone. Thus, area TE is critical for rapid visual object recognition, and detailed image-level visual information can reach area TE via a route other than through TEO.

Chemogenetic Disconnection between the Orbitofrontal Cortex and the Rostromedial Caudate Nucleus Disrupts Motivational Control of Goal-Directed Action.

The orbitofrontal cortex (OFC) and its major downstream target within the basal ganglia-the rostromedial caudate nucleus (rmCD)-are involved in reward-value processing and goal-directed behavior. However, a causal contribution of the pathway linking these two structures to goal-directed behavior has not been established. Using the chemogenetic technology of designer receptors exclusively activated by designer drugs with a crossed inactivation design, we functionally and reversibly disrupted interactions between the OFC and rmCD in two male macaque monkeys. We injected an adeno-associated virus vector expressing an inhibitory designer receptor, hM4Di, into the OFC and contralateral rmCD, the expression of which was visualized in vivo by positron emission tomography and confirmed by postmortem immunohistochemistry. Functional disconnection of the OFC and rmCD resulted in a significant and reproducible loss of sensitivity to the cued reward value for goal-directed action. This decreased sensitivity was most prominent when monkeys had accumulated a certain amount of reward. These results provide causal evidence that the interaction between the OFC and the rmCD is needed for motivational control of action on the basis of the relative reward value and internal drive. This finding extends the current understanding of the physiological basis of psychiatric disorders in which goal-directed behavior is affected, such as obsessive-compulsive disorder.SIGNIFICANCE STATEMENT In daily life, we routinely adjust the speed and accuracy of our actions on the basis of the value of expected reward. Abnormalities in these kinds of motivational adjustments might be related to behaviors seen in psychiatric disorders such as obsessive-compulsive disorder. In the current study, we show that the connection from the orbitofrontal cortex to the rostromedial caudate nucleus is essential for motivational control of action in monkeys. This finding expands our knowledge about how the primate brain controls motivation and behavior and provides a particular insight into disorders like obsessive-compulsive disorder in which altered connectivity between the orbitofrontal cortex and the striatum has been implicated.

Comparing performance between a deep neural network and monkeys with bilateral removals of visual area TE in categorizing feature-ambiguous stimuli.

In the canonical view of visual processing the neural representation of complex objects emerges as visual information is integrated through a set of convergent, hierarchically organized processing stages, ending in the primate inferior temporal lobe. It seems reasonable to infer that visual perceptual categorization requires the integrity of anterior inferior temporal cortex (area TE). Many deep neural networks (DNNs) are structured to simulate the canonical view of hierarchical processing within the visual system. However, there are some discrepancies between DNNs and the primate brain. Here we evaluated the performance of a simulated hierarchical model of vision in discriminating the same categorization problems presented to monkeys with TE removals. The model was able to simulate the performance of monkeys with TE removals in the categorization task but performed poorly when challenged with visually degraded stimuli. We conclude that further development of the model is required to match the level of visual flexibility present in the monkey visual system.

Contributions of the Monkey Inferior Temporal Areas TE and TEO to Visual Categorization.

The ability to categorize images is thought to depend on neural processing within the ventral visual stream. Recently, we reported that after removal of architectonic area TE, the terminal region of the ventral stream, monkeys were still able to categorize images as cats or dogs moderately well. Here, we investigate the contribution of TEO, the architectonically defined region located one step earlier than area TE in the ventral stream. Bilateral removal of TEO caused only a mild impairment in categorization. However, combined TE + TEO removal was followed by a severe, long-lasting impairment in categorization. All of the monkeys tested, including those with combined TE + TEO removals, had normal low-level visual functions, such as visual acuity. These results support the conclusion that categorization based on visual similarity is processed in parallel in TE and TEO.

Curvilinear features are important for animate/inanimate categorization in macaques.

The current experiment investigated the extent to which perceptual categorization of animacy (i.e., the ability to discriminate animate and inanimate objects) is facilitated by image-based features that distinguish the two object categories. We show that, with nominal training, naïve macaques could classify a trial-unique set of 1000 novel images with high accuracy. To test whether image-based features that naturally differ between animate and inanimate objects, such as curvilinear and rectilinear information, contribute to the monkeys' accuracy, we created synthetic images using an algorithm that distorted the global shape of the original animate/inanimate images while maintaining their intermediate features (Portilla & Simoncelli, 2000). Performance on the synthesized images was significantly above chance and was predicted by the amount of curvilinear information in the images. Our results demonstrate that, without training, macaques can use an intermediate image feature, curvilinearity, to facilitate their categorization of animate and inanimate objects.

Mild Perceptual Categorization Deficits Follow Bilateral Removal of Anterior Inferior Temporal Cortex in Rhesus Monkeys.

In primates, visual recognition of complex objects depends on the inferior temporal lobe. By extension, categorizing visual stimuli based on similarity ought to depend on the integrity of the same area. We tested three monkeys before and after bilateral anterior inferior temporal cortex (area TE) removal. Although mildly impaired after the removals, they retained the ability to assign stimuli to previously learned categories, e.g., cats versus dogs, and human versus monkey faces, even with trial-unique exemplars. After the TE removals, they learned in one session to classify members from a new pair of categories, cars versus trucks, as quickly as they had learned the cats versus dogs before the removals. As with the dogs and cats, they generalized across trial-unique exemplars of cars and trucks. However, as seen in earlier studies, these monkeys with TE removals had difficulty learning to discriminate between two simple black and white stimuli. These results raise the possibility that TE is needed for memory of simple conjunctions of basic features, but that it plays only a small role in generalizing overall configural similarity across a large set of stimuli, such as would be needed for perceptual categorical assignment. SIGNIFICANCE STATEMENT: The process of seeing and recognizing objects is attributed to a set of sequentially connected brain regions stretching forward from the primary visual cortex through the temporal lobe to the anterior inferior temporal cortex, a region designated area TE. Area TE is considered the final stage for recognizing complex visual objects, e.g., faces. It has been assumed, but not tested directly, that this area would be critical for visual generalization, i.e., the ability to place objects such as cats and dogs into their correct categories. Here, we demonstrate that monkeys rapidly and seemingly effortlessly categorize large sets of complex images (cats vs dogs, cars vs trucks), surprisingly, even after removal of area TE, leaving a puzzle about how this generalization is done.

Comparative physiology and morphology of BLA-projecting NBM/SI cholinergic neurons in mouse and macaque.

Cholinergic projection neurons of the nucleus basalis and substantia innominata (NBM/SI) densely innervate the basolateral amygdala (BLA) and have been shown to contribute to the encoding of fundamental and life-threatening experiences. Given the vital importance of these circuits in the acquisition and retention of memories that are essential for survival in a changing environment, it is not surprising that the basic anatomical organization of the NBM/SI is well conserved across animal classes as diverse as teleost and mammal. What is not known is the extent to which the physiology and morphology of NBM/SI neurons have also been conserved. To address this issue, we made patch-clamp recordings from NBM/SI neurons in ex vivo slices of two widely divergent mammalian species, mouse and rhesus macaque, focusing our efforts on cholinergic neurons that project to the BLA. We then reconstructed most of these recorded neurons post hoc to characterize neuronal morphology. We found that rhesus macaque BLA-projecting cholinergic neurons were both more intrinsically excitable and less morphologically compact than their mouse homologs. Combining measurements of 18 physiological features and 13 morphological features, we illustrate the extent of the separation. Although macaque and mouse neurons both exhibited considerable within-group diversity and overlapped with each other on multiple individual metrics, a combined morpho-electric analysis demonstrates that they form two distinct neuronal classes. Given the shared purpose of the circuits in which these neurons participate, this finding raises questions about (and offers constraints on) how these distinct classes result in similar behavior.

Synthesis and preclinical evaluation of [11C]uPSEM792 for PSAM4-GlyR based chemogenetics.

Chemogenetic tools are designed to control neuronal signaling. These tools have the potential to contribute to the understanding of neuropsychiatric disorders and to the development of new treatments. One such chemogenetic technology comprises modified Pharmacologically Selective Actuator Modules (PSAMs) paired with Pharmacologically Selective Effector Molecules (PSEMs). PSAMs are receptors with ligand-binding domains that have been modified to interact only with a specific small-molecule agonist, designated a PSEM. PSAM4 is a triple mutant PSAM derived from the α7 nicotinic receptor (α7L131G,Q139L,Y217F). Although having no constitutive activity as a ligand-gated ion channel, PSAM4 has been coupled to the serotonin 5-HT3 receptor (5-HT3R) and to the glycine receptor (GlyR). Treatment with the partner PSEM to activate PSAM4-5-HT3 or PSAM4-GlyR, causes neuronal activation or silencing, respectively. A suitably designed radioligand may enable selective visualization of the expression and location of PSAMs with positron emission tomography (PET). Here, we evaluated uPSEM792, an ultrapotent PSEM for PSAM4-GlyR, as a possible lead for PET radioligand development. We labeled uPSEM792 with the positron-emitter, carbon-11 (t1/2 = 20.4 min), in high radiochemical yield by treating a protected precursor with [11C]iodomethane followed by base deprotection. PET experiments with [11C]uPSEM792 in rodents and in a monkey transduced with PSAM4-GlyR showed low peak radioactivity uptake in brain. This low uptake was probably due to high polarity of the radioligand, as evidenced by physicochemical measurements, and to the vulnerability of the radioligand to efflux transport at the blood-brain barrier. These findings can inform the design of a more effective PSAM4 based PET radioligand, based on the uPSEM792 chemotype.

Distinct roles of monkey OFC-subcortical pathways in adaptive behavior.

Primates must adapt to changing environments by optimizing their behavior to make beneficial choices. At the core of adaptive behavior is the orbitofrontal cortex (OFC) of the brain, which updates choice value through direct experience or knowledge-based inference. Here, we identify distinct neural circuitry underlying these two separate abilities. We designed two behavioral tasks in which two male macaque monkeys updated the values of certain items, either by directly experiencing changes in stimulus-reward associations, or by inferring the value of unexperienced items based on the task's rules. Chemogenetic silencing of bilateral OFC combined with mathematical model-fitting analysis revealed that monkey OFC is involved in updating item value based on both experience and inference. In vivo imaging of chemogenetic receptors by positron emission tomography allowed us to map projections from the OFC to the rostromedial caudate nucleus (rmCD) and the medial part of the mediodorsal thalamus (MDm). Chemogenetic silencing of the OFC-rmCD pathway impaired experience-based value updating, while silencing the OFC-MDm pathway impaired inference-based value updating. Our results thus demonstrate dissociable contributions of distinct OFC projections to different behavioral strategies, and provide new insights into the neural basis of value-based adaptive decision-making in primates.

Divergent opioid-mediated suppression of inhibition between hippocampus and neocortex across species and development.

Opioid receptors within the CNS regulate pain sensation and mood and are key targets for drugs of abuse. Within the adult rodent hippocampus (HPC), µ-opioid receptor agonists suppress inhibitory parvalbumin-expressing interneurons (PV-INs), thus disinhibiting the circuit. However, it is uncertain if this disinhibitory motif is conserved in other cortical regions, species, or across development. We observed that PV-IN mediated inhibition is robustly suppressed by opioids in HPC but not neocortex in mice and nonhuman primates, with spontaneous inhibitory tone in resected human tissue also following a consistent dichotomy. This hippocampal disinhibitory motif was established in early development when immature PV-INs and opioids already influence primordial network rhythmogenesis. Acute opioid-mediated modulation was partially occluded with morphine pretreatment, with implications for the effects of opioids on hippocampal network activity during circuit maturation as well as learning and memory. Together, these findings demonstrate that PV-INs exhibit a divergence in opioid sensitivity across brain regions that is remarkably conserved across evolution and highlights the underappreciated role of opioids acting through immature PV-INs in shaping hippocampal development.

An enhancer-AAV toolbox to target and manipulate distinct interneuron subtypes.

In recent years, we and others have identified a number of enhancers that, when incorporated into rAAV vectors, can restrict the transgene expression to particular neuronal populations. Yet, viral tools to access and manipulate fine neuronal subtypes are still limited. Here, we performed systematic analysis of single cell genomic data to identify enhancer candidates for each of the cortical interneuron subtypes. We established a set of enhancer-AAV tools that are highly specific for distinct cortical interneuron populations and striatal cholinergic neurons. These enhancers, when used in the context of different effectors, can target (fluorescent proteins), observe activity (GCaMP) and manipulate (opto- or chemo-genetics) specific neuronal subtypes. We also validated our enhancer-AAV tools across species. Thus, we provide the field with a powerful set of tools to study neural circuits and functions and to develop precise and targeted therapy.

Inconsistencies between human and macaque lesion data can be resolved with a stimulus-computable model of the ventral visual stream.

Decades of neuroscientific research has sought to understand medial temporal lobe (MTL) involvement in perception. Apparent inconsistencies in the literature have led to competing interpretations of the available evidence; critically, findings from human participants with naturally occurring MTL damage appear to be inconsistent with data from monkeys with surgical lesions. Here, we leverage a 'stimulus-computable' proxy for the primate ventral visual stream (VVS), which enables us to formally evaluate perceptual demands across stimulus sets, experiments, and species. With this modeling framework, we analyze a series of experiments administered to monkeys with surgical, bilateral damage to perirhinal cortex (PRC), an MTL structure implicated in visual object perception. Across experiments, PRC-lesioned subjects showed no impairment on perceptual tasks; this originally led us(Eldridge et al., 2018) to conclude that PRC is not involved in perception. Here, we find that a 'VVS-like' model predicts both PRC-intact and -lesioned choice behaviors, suggesting that a linear readout of the VVS should be sufficient for performance on these tasks. Evaluating these computational results alongside findings from human experiments, we suggest that results from (Eldridge et al., 2018) alone cannot be used as evidence against PRC involvement in perception. These data indicate that experimental findings from human and non-human primates are consistent. As such, what appeared to be discrepancies between species was in fact due to reliance on informal accounts of perceptual processing.

Surgical Procedure for Implantation of Opto-Array in Nonhuman Primates.

Optogenetics allows precise temporal control of neuronal activity in the brain. Engineered viral vectors are routinely used to transduce neurons with light-sensitive opsins. However, reliable virus injection and light delivery in animals with large brains, such as nonhuman primates, has proven challenging. The Opto-Array is a novel yet simple device that is used to deliver light to extended regions of the cortex surface for high-throughput behavioral optogenetics in large brains. Here we present protocols for surgical delivery of virus (Basic Protocol 1) and implantation of the Opto-Array (Basic Protocol 2) in two separate surgeries in a rhesus monkey's inferior temporal cortex. As a proof of concept, we measured the behavioral performance of an animal detecting cortical optogenetic stimulations (Basic Protocol 3) with different illumination power and duration using the Opto-Array. The animal was able to detect the optogenetic stimulation for all tested illumination powers and durations. Regression analysis also showed both power and duration of illumination significantly modulate the detectability of the optogenetic stimulation. The outcome of this approach is superior to the standard practice of injecting and recording through a chamber for large areas of the cortex surface. Moreover, the chronic nature of the Opto-Array allows perturbation of neuronal activity of the same site across multiple sessions because it is highly stable; thus, data can be pooled over months. The detailed surgical method presented here makes it possible to use optogenetics to modulate neuronal activity across large regions of the cortex surface in the nonhuman primate brain. This method also lays the groundwork for future attempts to use optogenetics to restore vision in humans. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Virus injection surgery Basic Protocol 2: Opto-Array implantation surgery Basic Protocol 3: Cortical Perturbation Detection (CPD) task behavioral training.

PET reporter systems for the brain.

Positron emission tomography (PET) can be used as a noninvasive method to longitudinally monitor and quantify the expression of proteins in the brain in vivo. It can be used to monitor changes in biomarkers of mental health disorders, and to assess therapeutic interventions such as stem cell and molecular genetic therapies. The utility of PET monitoring depends on the availability of a radiotracer with good central nervous system (CNS) penetration and high selectivity for the target protein. This review evaluates existing methods for the visualization of reporter proteins and/or protein function using PET imaging, focusing on engineered systems, and discusses possible approaches for future success in the development of high-sensitivity and high-specificity PET reporter systems for the brain.

Image-dependence of the detectability of optogenetic stimulation in macaque inferotemporal cortex.

Artificial activation of neurons in early visual areas induces perception of simple visual flashes.1,2 Accordingly, stimulation in high-level visual cortices is expected to induce perception of complex features.3,4 However, results from studies in human patients challenge this expectation. Stimulation rarely induces any detectable visual event, and never a complex one, in human subjects with closed eyes.2 Stimulation of the face-selective cortex in a human patient led to remarkable hallucinations only while the subject was looking at faces.5 In contrast, stimulations of color- and face-selective sites evoke notable hallucinations independent of the object being viewed.6 These anecdotal observations suggest that stimulation of high-level visual cortex can evoke perception of complex visual features, but these effects depend on the availability and content of visual input. In this study, we introduce a novel psychophysical task to systematically investigate characteristics of the perceptual events evoked by optogenetic stimulation of macaque inferior temporal (IT) cortex. We trained macaque monkeys to detect and report optogenetic impulses delivered to their IT cortices7,8,9 while holding fixation on object images. In a series of experiments, we show that detection of cortical stimulation is highly dependent on the choice of images presented to the eyes and it is most difficult when fixating on a blank screen. These findings suggest that optogenetic stimulation of high-level visual cortex results in easily detectable distortions of the concurrent contents of vision.

Efficient viral expression of a chemogenetic receptor in the old-world monkey amygdala.

Genetically encoded synthetic receptors, such as the chemogenetic and optogenetic proteins, are powerful tools for functional brain studies in animals. In the primate brain, with its comparatively large, intricate anatomical structures, it can be challenging to express transgenes, such as the hM4Di chemogenetic receptor, in a defined anatomical structure with high penetrance. Here, we compare parameters for lentivirus vector injections in the rhesus monkey amygdala. We find that four injections of 20 µl, infused at 0.5 µl/min, can achieve neuronal hM4Di expression in 50-100% of neurons within a 60 mm3 volume, without observable damage from overexpression. Increasing the number of hM4Di_CFP lentivirus injections to up to 12 sites per hemisphere, resulted in 30%-40% neuronal coverage of the overall amygdala volume, with coverage reaching 60% in some subnuclei. Manganese Chloride was mixed with lentivirus and used as an MRI marker to verify targeting accuracy and correct unsuccessful injections in these experiments. In a separate monkey we visualized, in vivo, viral expression of the hM4Di receptor protein in the amygdala, using Positron Emission Tomography. Together, these data show efficient and verifiable expression of a chemogenetic receptor in old-world monkey amygdala.

Unilateral caudate inactivation increases motor impulsivity in rhesus monkeys.

Impulsivity, the tendency to react quickly and without consideration of consequences, is correlated with asymmetry in the volume of the caudate nucleus in human patients. In this study, we sought to determine whether the induction of functional asymmetry in the caudate nucleus of monkeys would produce phenomenologically comparable behavior. We found that unilateral suppression of the ventral caudate nucleus increases impulsive behavior in rhesus monkeys. Impulsivity was modeled by the subjects' inability to maintain hold of a touch-sensitive bar until presentation of an imperative signal. Two methods were used to suppress activity in the caudate region. First, muscimol was locally infused. Second, a viral construct expressing the hM4Di DREADD (designer receptor exclusively activated by designer drug) was injected at the same site. Clozapine N-oxide and deschloroclozapine activate the DREADD to suppress neuronal activity. Both methods of suppression, pharmacological and chemogenetic, increased the rate of early bar releases, a behavior we interpret to indicate impulsivity. Thus, we demonstrate a causal relationship between caudate asymmetry and impulsivity.

Neural correlates of category learning in monkey inferior temporal cortex.

We trained two monkeys implanted with multi-electrode arrays to categorize natural images of cats and dogs, in order to observe changes in neural activity related to category learning. We recorded neural activity from area TE, which is required for normal learning of visual categories based on perceptual similarity. Neural activity during a passive viewing task was compared pre- and post-training. After the category training, the accuracy of abstract category decoding improved. Specifically, the proportion of single units with category selectivity increased, and units sustained their category-specific responses for longer. Visual category learning thus appears to enhance category separability in area TE by driving changes in the stimulus selectivity of individual neurons and by recruiting more units to the active network.