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.

Exploring strategy differences between humans and monkeys with recurrent neural networks.

Animal models are used to understand principles of human biology. Within cognitive neuroscience, non-human primates are considered the premier model for studying decision-making behaviors in which direct manipulation experiments are still possible. Some prominent studies have brought to light major discrepancies between monkey and human cognition, highlighting problems with unverified extrapolation from monkey to human. Here, we use a parallel model system-artificial neural networks (ANNs)-to investigate a well-established discrepancy identified between monkeys and humans with a working memory task, in which monkeys appear to use a recency-based strategy while humans use a target-selective strategy. We find that ANNs trained on the same task exhibit a progression of behavior from random behavior (untrained) to recency-like behavior (partially trained) and finally to selective behavior (further trained), suggesting monkeys and humans may occupy different points in the same overall learning progression. Surprisingly, what appears to be recency-like behavior in the ANN, is in fact an emergent non-recency-based property of the organization of the neural network's state space during its development through training. We find that explicit encouragement of recency behavior during training has a dual effect, not only causing an accentuated recency-like behavior, but also speeding up the learning process altogether, resulting in an efficient shaping mechanism to achieve the optimal strategy. Our results suggest a new explanation for the discrepency observed between monkeys and humans and reveal that what can appear to be a recency-based strategy in some cases may not be recency at all.

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.

RNAi and chemogenetic reporter co-regulation in primate striatal interneurons.

Using genetic tools to study the functional roles of molecularly specified neuronal populations in the primate brain is challenging, primarily because of specificity and verification of virus-mediated targeting. Here, we report a lentivirus-based system that helps improve specificity and verification by (a) targeting a selected molecular mechanism, (b) in vivo reporting of expression, and (c) allowing the option to independently silence all regional neural activity. Specifically, we modulate cholinergic signaling of striatal interneurons by shRNAmir and pair it with hM4Di_CFP, a chemogenetic receptor that can function as an in vivo and in situ reporter. Quantitative analyses by visual and deep-learning assisted methods show an inverse linear relation between hM4Di_CFP and ChAT protein expression for several shRNAmir constructs. This approach successfully applies shRNAmir to modulating gene expression in the primate brain and shows that hM4Di_CFP can act as a readout for this modulation.

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.

Temporal Coding of Reward Value in Monkey Ventral Striatal Tonically Active Neurons.

The rostromedioventral striatum is critical for behavior dependent on evaluating rewards. We asked what contribution tonically active neurons (TANs), the putative striatal cholinergic interneurons, make in coding reward value in this part of the striatum. Two female monkeys were given the option to accept or reject an offered reward in each trial, the value of which was signaled by a visual cue. Forty-five percent of the TANs use temporally modulated activity to encode information about discounted value. These responses were significantly better represented using principal component analysis than by just counting spikes. The temporal coding is straightforward: the spikes are distributed according to a sinusoidal envelope of activity that changes gain, ranging from positive to negative according to discounted value. Our results show that the information about the relative value of an offered reward is temporally encoded in neural spike trains of TANs. This temporal coding may allow well tuned, coordinated behavior to emerge.SIGNIFICANCE STATEMENT Ever since the discovery that neurons use trains of pulses to transmit information, it seemed self-evident that information would be encoded into the pattern of the spikes. However, there is not much evidence that spike patterns encode cognitive information. We find that a set of interneurons, the tonically active neurons (TANs) in monkeys' striatum, use temporal patterns of response to encode information about the discounted value of offered rewards. The code seems straightforward: a sinusoidal envelope that changes gain according to the discounted value of the offer, describes the rate of spiking across time. This temporal modulation may provide a means to synchronize these interneurons and the activity of other neural elements including principal output neurons.

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.

Multimodal Imaging for DREADD-Expressing Neurons in Living Brain and Their Application to Implantation of iPSC-Derived Neural Progenitors.

Chemogenetic manipulation of neuronal activities has been enabled by a designer receptor (designer receptor exclusively activated by designer drugs, DREADD) that is activated exclusively by clozapine-N-oxide (CNO). Here, we applied CNO as a functional reporter probe to positron emission tomography (PET) of DREADD in living brains. Mutant human M4 DREADD (hM4Di) expressed in transgenic (Tg) mouse neurons was visualized by PET with microdose [11C]CNO. Deactivation of DREADD-expressing neurons in these mice by nonradioactive CNO at a pharmacological dose could also be captured by arterial spin labeling MRI (ASL-MRI). Neural progenitors derived from hM4Di Tg-induced pluripotent stem cells were then implanted into WT mouse brains and neuronal differentiation of the grafts could be imaged by [11C]CNO-PET. Finally, ASL-MRI captured chemogenetic functional manipulation of the graft neurons. Our data provide the first demonstration of multimodal molecular/functional imaging of cells expressing a functional gene reporter in the brain, which would be translatable to humans for therapeutic gene transfers and cell replacements. SIGNIFICANCE STATEMENT: The present work provides the first successful demonstration of in vivo positron emission tomographic (PET) visualization of a chemogenetic designer receptor (designer receptor exclusively activated by designer drugs, DREADD) expressed in living brains. This technology has been applied to longitudinal PET reporter imaging of neuronal grafts differentiated from induced pluripotent stem cells. Differentiated from currently used reporter genes for neuroimaging, DREADD has also been available for functional manipulation of target cells, which could be visualized by functional magnetic resonance imaging (fMRI) in a real-time manner. Multimodal imaging with PET/fMRI enables the visualization of the differentiation of iPSC-derived neural progenitors into mature neurons and DREADD-mediated functional manipulation along the time course of the graft and is accordingly capable of fortifying the utility of stem cells in cell replacement therapies.

Humans and monkeys use different strategies to solve the same short-term memory tasks.

The neural mechanisms underlying human working memory are often inferred from studies using old-world monkeys. Humans use working memory to selectively memorize important information. We recently reported that monkeys do not seem to use selective memorization under experimental conditions that are common in monkey research, but less common in human research. Here we compare the performance of humans and monkeys under the same experimental conditions. Humans selectively remember important images whereas monkeys largely rely on recency information from nonselective memorization. Working memory studies in old-world monkeys must be interpreted cautiously when making inferences about the mechanisms underlying human working memory.

Sensitivity of locus ceruleus neurons to reward value for goal-directed actions.

The noradrenergic nucleus locus ceruleus (LC) is associated classically with arousal and attention. Recent data suggest that it might also play a role in motivation. To study how LC neuronal responses are related to motivational intensity, we recorded 121 single neurons from two monkeys while reward size (one, two, or four drops) and the manner of obtaining reward (passive vs active) were both manipulated. The monkeys received reward under three conditions: (1) releasing a bar when a visual target changed color; (2) passively holding a bar; or (3) touching and releasing a bar. In the first two conditions, a visual cue indicated the size of the upcoming reward, and, in the third, the reward was constant through each block of 25 trials. Performance levels and lipping intensity (an appetitive behavior) both showed that the monkeys' motivation in the task was related to the predicted reward size. In conditions 1 and 2, LC neurons were activated phasically in relation to cue onset, and this activation strengthened with increasing expected reward size. In conditions 1 and 3, LC neurons were activated before the bar-release action, and the activation weakened with increasing expected reward size but only in task 1. These effects evolved as monkeys progressed through behavioral sessions, because increasing fatigue and satiety presumably progressively decreased the value of the upcoming reward. These data indicate that LC neurons integrate motivationally relevant information: both external cues and internal drives. The LC might provide the impetus to act when the predicted outcome value is low.

Neural mechanisms underlying contextual dependency of subjective values: converging evidence from monkeys and humans.

A major challenge for decision theory is to account for the instability of expressed preferences across time and context. Such variability could arise from specific properties of the brain system used to assign subjective values. Growing evidence has identified the ventromedial prefrontal cortex (VMPFC) as a key node of the human brain valuation system. Here, we first replicate this observation with an fMRI study in humans showing that subjective values of painting pictures, as expressed in explicit pleasantness ratings, are specifically encoded in the VMPFC. We then establish a bridge with monkey electrophysiology, by comparing single-unit activity evoked by visual cues between the VMPFC and the orbitofrontal cortex. At the neural population level, expected reward magnitude was only encoded in the VMPFC, which also reflected subjective cue values, as expressed in Pavlovian appetitive responses. In addition, we demonstrate in both species that the additive effect of prestimulus activity on evoked activity has a significant impact on subjective values. In monkeys, the factor dominating prestimulus VMPFC activity was trial number, which likely indexed variations in internal dispositions related to fatigue or satiety. In humans, prestimulus VMPFC activity was externally manipulated through changes in the musical context, which induced a systematic bias in subjective values. Thus, the apparent stochasticity of preferences might relate to the VMPFC automatically aggregating the values of contextual features, which would bias subsequent valuation because of temporal autocorrelation in neural activity.

Monkeys rely on recency of stimulus repetition when solving short-term memory tasks.

Seven monkeys performed variants of two short-term memory tasks that others have used to differentiate between selective and nonselective memory mechanisms. The first task was to view a list of sequentially presented images and identify whether a test matched any image from the list, but not a distractor from a preceding list. Performance was best when the test matched the most recently presented image. Response rates depended linearly on recency of repetition whether the test matched a sample from the current list or a distractor from a preceding list, suggesting nonselective memorization of all images viewed instead of just the sample images. The second task was to remember just the first image in a list selectively and ignore subsequent distractors. False alarms occurred frequently when the test matched a distractor presented near the beginning of the sequence. In a pilot experiment, response rates depended linearly on recency of repetition irrespective of whether the test matched the first image or a distractor, again suggesting nonselective memorization of all images instead of just the first image. Modification of the second task improved recognition of the first image, but did not abolish use of recency. Monkeys appear to perform nonspatial visual short-term memory tasks often (or exclusively) using a single, nonselective, memory mechanism that conveys the recency of stimulus repetition.

Stochastic reinforcement benefits skill acquisition.

Learning complex skills is driven by reinforcement, which facilitates both online within-session gains and retention of the acquired skills. Yet, in ecologically relevant situations, skills are often acquired when mapping between actions and rewarding outcomes is unknown to the learning agent, resulting in reinforcement schedules of a stochastic nature. Here we trained subjects on a visuomotor learning task, comparing reinforcement schedules with higher, lower, or no stochasticity. Training under higher levels of stochastic reinforcement benefited skill acquisition, enhancing both online gains and long-term retention. These findings indicate that the enhancing effects of reinforcement on skill acquisition depend on reinforcement schedules.

Neurons in monkey dorsal raphe nucleus code beginning and progress of step-by-step schedule, reward expectation, and amount of reward outcome in the reward schedule task.

The dorsal raphe nucleus is the major source of serotonin in the brain. It is connected to brain regions related to reward processing, and the neurons show activity related to predicted reward outcome. Clinical observations also suggest that it is important in maintaining alertness and its apparent role in addiction seems to be related to reward processing. Here, we examined whether the neurons in dorsal raphe carry signals about reward outcome and task progress during multitrial schedules. We recorded from 98 single neurons in dorsal raphe of two monkeys. The monkeys perform one, two, or three visual discrimination trials (schedule), obtaining one, two, or three drops of liquid. In the valid cue condition, the length and brightness of a visual cue indicated schedule progress and reward amount, respectively. In the random cue condition, the visual cue was randomly presented with respect to schedule length and reward amount. We found information encoded about (1) schedule onset, (2) reward expectation, (3) reward outcome, and (4) reward amount in the mean firing rates. Information theoretic analysis showed that the temporal variation of the neuronal responses contained additional information related to the progress of the schedule toward the reward rather than only discriminating schedule onset or reward/no reward. When considered in light of all that is known about the raphe in anatomy, physiology, and behavior, the rich encoding about both task progress and predicted reward outcome makes the raphe a strong candidate for providing signals throughout the brain to coordinate persistent goal-seeking behavior.

Interaction between orbital prefrontal and rhinal cortex is required for normal estimates of expected value.

Predicting and valuing potential rewards requires integrating sensory, associative, and contextual information with subjective reward preferences. Previous work has identified regions in the prefrontal cortex and medial temporal lobe believed to be important for each of these functions. For example, activity in the orbital prefrontal cortex (PFo) encodes the specific sensory properties of and preferences for rewards, while activity in the rhinal cortex (Rh) encodes stimulus-stimulus and stimulus-reward associations. Lesions of either structure impair the ability to use visual cues or the history of previous reinforcement to value expected rewards. These areas are linked via reciprocal connections, suggesting it might be their interaction that is critical for estimating expected value. To test this hypothesis, we interrupted direct, intra-hemispheric PFo-Rh interaction in monkeys by performing crossed unilateral ablations of these regions (functional disconnection). We asked whether this circuit is crucial primarily for cue-reward association or for estimating expected value per se, by testing these monkeys, as well as intact controls, on tasks in which expected value was either visually cued or had to be inferred from block-wise changes in reward size in uncued trials. Functional disconnection significantly affected performance in both tasks. Specifically, monkeys with functional disconnection showed less of a difference in error rates and reaction times across reward sizes, in some cases behaving as if they expected rewards to be of equal magnitude. These results support a model whereby information about rewards signaled in PFo is combined with associative and contextual information signaled within Rh to estimate expected value.

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.

Intersection of reward and memory in monkey rhinal cortex.

In humans and other animals, the vigor with which a reward is pursued depends on its desirability, that is, on the reward's predicted value. Predicted value is generally context-dependent, varying according to the value of rewards obtained in the recent and distant past. Signals related to reward prediction and valuation are believed to be encoded in a circuit centered around midbrain dopamine neurons and their targets in the prefrontal cortex and basal ganglia. Notably absent from this hypothesized reward pathway are dopaminergic targets in the medial temporal lobe. Here we show that a key part of the medial temporal lobe memory system previously reported to be important for sensory mnemonic and perceptual processing, the rhinal cortex (Rh), is required for using memories of previous reward values to predict the value of forthcoming rewards. We tested monkeys with bilateral Rh lesions on a task in which reward size varied across blocks of uncued trials. In this experiment, the only cues for predicting current reward value are the sizes of rewards delivered in previous blocks. Unexpectedly, monkeys with Rh ablations, but not intact controls, were insensitive to differences in predicted reward, responding as if they expected all rewards to be of equal magnitude. Thus, it appears that Rh is critical for using memory of previous rewards to predict the value of forthcoming rewards. These results are in agreement with accumulating evidence that Rh is critical for establishing the relationships between temporally interleaved events, which is a key element of episodic memory.