Advancing clinical development for neuronopathic Hunter syndrome through a quantitatively-driven reverse translation framework.

A quantitatively-driven evaluation of existing clinical data and associated knowledge to accelerate drug discovery and development is a highly valuable approach across therapeutic areas, but remains underutilized. This is especially the case for rare diseases for which development is particularly challenging. The current work outlines an organizational framework to support a quantitatively-based reverse translation approach to clinical development. This approach was applied to characterize predictors of the trajectory of cognition in Hunter syndrome (Mucopolysaccharidosis Type II; MPS-II), a rare X-linked lysosomal storage disorder, highly heterogeneous in its course. Specifically, we considered ways to refine target populations based on age, cognitive status, and biomarkers, that is, cerebrospinal fluid glycosaminoglycans (GAG), at trial entry. Data from a total of 138 subjects (age range 2.5 to 10.1 years) from Takeda-sponsored internal studies and external natural history studies in MPS-II were included. Quantitative analyses using mixed-effects models were performed to characterize the relationships between neurocognitive outcomes and potential indicators of disease progression. Results revealed a specific trajectory in cognitive development across age with an initial progressive phase, followed by a plateau between 4 and 8 years and then a variable declining phase. Additionally, results suggest a faster decline in cognition among subjects with lower cognitive scores or with higher cerebrospinal fluid GAG at enrollment. These results support differences in the neurocognitive course of MPS-II between distinct groups of patients based on age, cognitive function, and biomarker status at enrollment. These differences should be considered when designing future clinical trials.

Seizure reduction in TSC2-mutant mouse model by an mTOR catalytic inhibitor.

OBJECTIVE: Tuberous sclerosis complex (TSC) is a neurodevelopmental disorder caused by autosomal-dominant pathogenic variants in either the TSC1 or TSC2 gene, and it is characterized by hamartomas in multiple organs, such as skin, kidney, lung, and brain. These changes can result in epilepsy, learning disabilities, and behavioral complications, among others. The mechanistic link between TSC and the mechanistic target of the rapamycin (mTOR) pathway is well established, thus mTOR inhibitors can potentially be used to treat the clinical manifestations of the disorder, including epilepsy. METHODS: In this study, we tested the efficacy of a novel mTOR catalytic inhibitor (here named Tool Compound 1 or TC1) previously reported to be more brain-penetrant compared with other mTOR inhibitors. Using a well-characterized hypomorphic Tsc2 mouse model, which displays a translationally relevant seizure phenotype, we tested the efficacy of TC1. RESULTS: Our results show that chronic treatment with this novel mTOR catalytic inhibitor (TC1), which affects both the mTORC1 and mTORC2 signaling complexes, reduces seizure burden, and extends the survival of Tsc2 hypomorphic mice, restoring species typical weight gain over development. INTERPRETATION: Novel mTOR catalytic inhibitor TC1 exhibits a promising therapeutic option in the treatment of TSC.

GENE TARGET: A framework for evaluating Mendelian neurodevelopmental disorders for gene therapy.

Interest in gene-based therapies for neurodevelopmental disorders is increasing exponentially, driven by the rise in recognition of underlying genetic etiology, progress in genomic technology, and recent proof of concept in several disorders. The current prioritization of one genetic disorder over another for development of therapies is driven by competing interests of pharmaceutical companies, advocacy groups, and academic scientists. Although these are all valid perspectives, a consolidated framework will facilitate more efficient and rational gene therapy development. Here we outline features of Mendelian neurodevelopmental disorders that warrant consideration when determining suitability for gene therapy. These features fit into four broad domains: genetics, preclinical validation, clinical considerations, and ethics. We propose a simple mnemonic, GENE TARGET, to remember these features and illustrate how they could be scored using a preliminary scoring rubric. In this suggested rubric, for a given disorder, scores for each feature may be added up to a composite GENE TARGET suitability (GTS) score. In addition to proposing a systematic method to evaluate and compare disorders, our framework helps identify gaps in the translational pipeline for a given disorder, which can inform prioritization of future research efforts.

Shifted phase of EEG cross-frequency coupling in individuals with Phelan-McDermid syndrome.

BACKGROUND: Phelan-McDermid Syndrome (PMS) is a rare condition caused by deletion or mutation of the SHANK3 gene. Individuals with PMS frequently present with intellectual disability, autism spectrum disorder, and other neurodevelopmental challenges. Electroencephalography (EEG) can provide a window into network-level function in PMS. METHODS: Here, we analyze EEG data collected across multiple sites in individuals with PMS (n = 26) and typically developing individuals (n = 15). We quantify oscillatory power, alpha-gamma phase-amplitude coupling strength, and phase bias, a measure of the phase of cross frequency coupling thought to reflect the balance of feedforward (bottom-up) and feedback (top-down) activity. RESULTS: We find individuals with PMS display increased alpha-gamma phase bias (U = 3.841, p < 0.0005), predominantly over posterior electrodes. Most individuals with PMS demonstrate positive overall phase bias while most typically developing individuals demonstrate negative overall phase bias. Among individuals with PMS, strength of alpha-gamma phase-amplitude coupling was associated with Sameness, Ritualistic, and Compulsive behaviors as measured by the Repetitive Behavior Scales-Revised (Beta = 0.545, p = 0.011). CONCLUSIONS: Increased phase bias suggests potential circuit-level mechanisms underlying phenotype in PMS, offering opportunities for back-translation of findings into animal models and targeting in clinical trials.

Phenotypic Screen with TSC-Deficient Neurons Reveals Heat-Shock Machinery as a Druggable Pathway for mTORC1 and Reduced Cilia.

Tuberous sclerosis complex (TSC) is a neurogenetic disorder that leads to elevated mechanistic targeting of rapamycin complex 1 (mTORC1) activity. Cilia can be affected by mTORC1 signaling, and ciliary deficits are associated with neurodevelopmental disorders. Here, we examine whether neuronal cilia are affected in TSC. We show that cortical tubers from TSC patients and mutant mouse brains have fewer cilia. Using high-content image-based assays, we demonstrate that mTORC1 activity inversely correlates with ciliation in TSC1/2-deficient neurons. To investigate the mechanistic relationship between mTORC1 and cilia, we perform a phenotypic screen for mTORC1 inhibitors with TSC1/2-deficient neurons. We identify inhibitors of the heat shock protein 90 (Hsp90) that suppress mTORC1 through regulation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling. Pharmacological inhibition of Hsp90 rescues ciliation through downregulation of Hsp27. Our study uncovers the heat-shock machinery as a druggable signaling node to restore mTORC1 activity and cilia due to loss of TSC1/2, and it provides broadly applicable platforms for studying TSC-related neuronal dysfunction.

Tau: A Novel Entry Point for mTOR-Based Treatments in Autism Spectrum Disorder?

Dysregulation of the PI3K/Akt/mTOR pathway has become a point of convergence in autism spectrum disorder (ASD). In this issue of Neuron, Tai et al. (2020) identify a therapeutic role for tau reduction in downregulating this pathway and ameliorating ASD-like symptoms.

Auditory Processing of Speech and Tones in Children With Tuberous Sclerosis Complex.

Individuals with Tuberous Sclerosis Complex (TSC) have atypical white matter integrity and neural connectivity in the brain, including language pathways. To explore functional activity associated with auditory and language processing in individuals with TSC, we used electroencephalography (EEG) to examine basic auditory correlates of detection (P1, N2, N4) and discrimination (mismatch negativity, MMN) of speech and non-speech stimuli for children with TSC and age- and sex-matched typically developing (TD) children. Children with TSC (TSC group) and without TSC (typically developing, TD group) participated in an auditory MMN paradigm containing two blocks of vowels (/a/and/u/) and two blocks of tones (800 Hz and 400 Hz). Continuous EEG data were collected. Multivariate pattern analysis (MVPA) was used to explore functional specificity of neural auditory processing. Speech-specific P1, N2, and N4 waveform components of the auditory evoked potential (AEP) were compared, and the mismatch response was calculated for both speech and tones. MVPA showed that the TD group, but not the TSC group, demonstrated above-chance pairwise decoding between speech and tones. The AEP component analysis suggested that while the TD group had an increased P1 amplitude in response to vowels compared to tones, the TSC group did not show this enhanced response to vowels. Additionally, the TD group had a greater N2 amplitude in response to vowels, but not tones, compared to the TSC group. The TSC group also demonstrated a longer N4 latency to vowels compared to tones, which was not seen in the TD group. No group differences were observed in the MMN response. In this study we identified features of the auditory response to speech sounds, but not acoustically matched tones, which differentiate children with TSC from TD children.

A framework for the investigation of rare genetic disorders in neuropsychiatry.

De novo and inherited rare genetic disorders (RGDs) are a major cause of human morbidity, frequently involving neuropsychiatric symptoms. Recent advances in genomic technologies and data sharing have revolutionized the identification and diagnosis of RGDs, presenting an opportunity to elucidate the mechanisms underlying neuropsychiatric disorders by investigating the pathophysiology of high-penetrance genetic risk factors. Here we seek out the best path forward for achieving these goals. We think future research will require consistent approaches across multiple RGDs and developmental stages, involving both the characterization of shared neuropsychiatric dimensions in humans and the identification of neurobiological commonalities in model systems. A coordinated and concerted effort across patients, families, researchers, clinicians and institutions, including rapid and broad sharing of data, is now needed to translate these discoveries into urgently needed therapies.

A unified circuit for social behavior.

Recent advances in circuit manipulation technologies have enabled the association of distinct neural circuits with complex social behaviors. The brain areas identified through historical anatomical characterizations as mediators of sexual and parental behaviors can now be functionally linked to adult social behaviors within a unified circuit. In vivo electrophysiology, optogenetics and chemogenetics have been used to follow the processing of social sensory stimuli from perception by the olfactory system to valence detection by the amygdala and mesolimbic dopamine system to integration by the cerebral and cerebellar cortices under modulation of hypothalamic neuropeptides. Further, these techniques have been able to identify the distinct functional changes induced by social as opposed to non-social stimuli. Together this evidence suggests that there is a distinct, functionally coupled circuit that is selectively activated by social stimuli. A unified social circuit provides a new framework against which synaptopathic autism related mutations can be considered and novel pharmacotherapeutic strategies can be developed.

Electrographic spikes are common in wildtype mice.

High-voltage rhythmic electroencephalographic (EEG) spikes have been recorded in wildtype (WT) rats during periods of light slow-wave sleep and passive wakefulness. The source of this activity is unclear but has been attributed to either an inherent form of absence epilepsy or a normal feature of rodent sleep EEG. In contrast, little is known about epileptiform spikes in WT mice. We thus characterize and quantify epileptiform discharges in WT mice for the first time. Thirty-six male WT C57 mice with 24-h wireless telemetry video-EEG recordings were manually scored by blinded reviewers to mark individual spikes and spike trains. Epileptiform spikes were detected in 100% of the recorded WT mice, and spike trains of at least three spikes were recorded in 90% of mice. The spikes were more frequent during the day than at night and were inversely correlated to each animal's locomotor activity. However, the discharges were not absent during active nighttime periods. These discharges may indicate a baseline tendency toward epileptic seizures or perhaps are benign variants of normal rodent background EEG. Nevertheless, a better understanding of baseline WT EEG activity will aid in differentiating pathological and normal EEG activity in mouse epilepsy models.

The Way Forward for Mechanism-Based Therapeutics in Genetically Defined Neurodevelopmental Disorders.

Rare genetically defined neurodevelopmental disorders with increased risk of autism have recently become an entry point for autism-related drug discovery. Through exploration of downstream effects of the pathological mutations, specific mechanistic pathways have been identified as dysregulated. The identification of shared mechanisms across forms of autism opens the door for the development of novel "mechanism-based therapeutics." However, confidence in the therapeutic mechanism does not diminish the need for well-designed clinical trials.

Hyperactivity and Hypermotivation Associated With Increased Striatal mGluR1 Signaling in a Shank2 Rat Model of Autism.

Mutations in the SHANK family of genes have been consistently identified in genetic and genomic screens of autism spectrum disorder (ASD). The functional overlap of SHANK with several other ASD-associated genes suggests synaptic dysfunction as a convergent mechanism of pathophysiology in ASD. Although many ASD-related mutations result in alterations to synaptic function, the nature of those dysfunctions and the consequential behavioral manifestations are highly variable when expressed in genetic mouse models. To investigate the phylogenetic conservation of phenotypes resultant of Shank2 loss-of-function in a translationally relevant animal model, we generated and characterized a novel transgenic rat with a targeted mutation of the Shank2 gene, enabling an evaluation of gene-associated phenotypes, the elucidation of complex behavioral phenotypes, and the characterization of potential translational biomarkers. The Shank2 loss-of-function mutation resulted in a notable phenotype of hyperactivity encompassing hypermotivation, increased locomotion, and repetitive behaviors. Mutant rats also expressed deficits in social behavior throughout development and in the acquisition of operant tasks. The hyperactive phenotype was associated with an upregulation of mGluR1 expression, increased dendritic branching, and enhanced long-term depression (LTD) in the striatum but opposing morphological and cellular alterations in the hippocampus (HP). Administration of the mGluR1 antagonist JNJ16259685 selectively normalized the expression of striatally mediated repetitive behaviors and physiology but had no effect on social deficits. Finally, Shank2 mutant animals also exhibited alterations in electroencephalography (EEG) spectral power and event-related potentials, which may serve as translatable EEG biomarkers of synaptopathic alterations. Our results show a novel hypermotivation phenotype that is unique to the rat model of Shank2 dysfunction, in addition to the traditional hyperactive and repetitive behaviors observed in mouse models. The hypermotivated and hyperactive phenotype is associated with striatal dysfunction, which should be explored further as a targetable mechanism for impairment in ASD.

mGluR5 Modulation of Behavioral and Epileptic Phenotypes in a Mouse Model of Tuberous Sclerosis Complex.

Drugs targeting metabotropic glutamate receptor 5 (mGluR5) have therapeutic potential in autism spectrum disorders (ASD), including tuberous sclerosis complex (TSC). The question whether inhibition or potentiation of mGluR5 could be beneficial depends, among other factors, on the specific indication. To facilitate the development of mGluR5 treatment strategies, we tested the therapeutic utility of mGluR5 negative and positive allosteric modulators (an mGluR5 NAM and PAM) for TSC, using a mutant mouse model with neuronal loss of Tsc2 that demonstrates disease-related phenotypes, including behavioral symptoms of ASD and epilepsy. This model uniquely enables the in vivo characterization and rescue of the electrographic seizures associated with TSC. We demonstrate that inhibition of mGluR5 corrects hyperactivity, seizures, and elevated de novo synaptic protein synthesis. Conversely, positive allosteric modulation of mGluR5 results in the exacerbation of hyperactivity and epileptic phenotypes. The data suggest a meaningful therapeutic potential for mGluR5 NAMs in TSC, which warrants clinical exploration and the continued development of mGluR5 therapies.

Replicable in vivo physiological and behavioral phenotypes of the Shank3B null mutant mouse model of autism.

BACKGROUND: Autism spectrum disorder (ASD) is a clinically and biologically heterogeneous condition characterized by social, repetitive, and sensory behavioral abnormalities. No treatments are approved for the core diagnostic symptoms of ASD. To enable the earliest stages of therapeutic discovery and development for ASD, robust and reproducible behavioral phenotypes and biological markers are essential to establish in preclinical animal models. The goal of this study was to identify electroencephalographic (EEG) and behavioral phenotypes that are replicable between independent cohorts in a mouse model of ASD. The larger goal of our strategy is to empower the preclinical biomedical ASD research field by generating robust and reproducible behavioral and physiological phenotypes in animal models of ASD, for the characterization of mechanistic underpinnings of ASD-relevant phenotypes, and to ensure reliability for the discovery of novel therapeutics. Genetic disruption of the SHANK3 gene, a scaffolding protein involved in the stability of the postsynaptic density in excitatory synapses, is thought to be responsible for a relatively large number of cases of ASD. Therefore, we have thoroughly characterized the robustness of ASD-relevant behavioral phenotypes in two cohorts, and for the first time quantified translational EEG activity in Shank3B null mutant mice. METHODS: In vivo physiology and behavioral assays were conducted in two independently bred and tested full cohorts of Shank3B null mutant (Shank3B KO) and wildtype littermate control (WT) mice. EEG was recorded via wireless implanted telemeters for 7 days of baseline followed by 20 min of recording following pentylenetetrazol (PTZ) challenge. Behaviors relevant to the diagnostic and associated symptoms of ASD were tested on a battery of established behavioral tests. Assays were designed to reproduce and expand on the original behavioral characterization of Shank3B KO mice. Two or more corroborative tests were conducted within each behavioral domain, including social, repetitive, cognitive, anxiety-related, sensory, and motor categories of assays. RESULTS: Relative to WT mice, Shank3B KO mice displayed a dramatic resistance to PTZ seizure induction and an enhancement of gamma band oscillatory EEG activity indicative of enhanced inhibitory tone. These findings replicated in two separate cohorts. Behaviorally, Shank3B KO mice exhibited repetitive grooming, deficits in aspects of reciprocal social interactions and vocalizations, and reduced open field activity, as well as variable deficits in sensory responses, anxiety-related behaviors, learning and memory. CONCLUSIONS: Robust animal models and quantitative, replicable biomarkers of neural dysfunction are needed to decrease risk and enable successful drug discovery and development for ASD and other neurodevelopmental disorders. Complementary to the replicated behavioral phenotypes of the Shank3B mutant mouse is the new identification of a robust, translational in vivo neurophysiological phenotype. Our findings provide strong evidence for robustness and replicability of key translational phenotypes in Shank3B mutant mice and support the usefulness of this mouse model of ASD for therapeutic discovery.

Translational use of event-related potentials to assess circuit integrity in ASD.

Deficits in social cognition are the defining characteristic of autism spectrum disorder (ASD). Social cognition requires the integration of several neural circuits in a time-sensitive fashion, so impairments in social interactions could arise as a result of alterations in network connectivity. Electroencephalography (EEG) has revealed abnormalities in event related potentials (ERPs) evoked by auditory and visual sensory stimuli in humans with ASD, indicating disruption of neural connectivity. Similar abnormalities in sensory-evoked ERPs have been observed in animal models of ASD, suggesting that ERPs have the potential to provide a translational biomarker of the disorder. People with ASD also have abnormal ERPs in response to auditory and visual social stimuli, demonstrating functional disruption of the social circuit. To assess the integrity of the social circuit and characterize biomarkers of circuit dysfunction, novel EEG paradigms that use social stimuli to induce ERPs should be developed for use in animal models. The identification of a socially-relevant ERP that is consistent in animal models and humans would facilitate the development of pharmacological treatment strategies for the social impairments in ASD and other neuropsychiatric disorders.

Peripheral Administration of a Long-Acting Peptide Oxytocin Receptor Agonist Inhibits Fear-Induced Freezing.

Oxytocin (OT) modulates the expression of social and emotional behaviors and consequently has been proposed as a pharmacologic treatment of psychiatric diseases, including autism spectrum disorders and schizophrenia; however, endogenous OT has a short half-life in plasma and poor permeability across the blood-brain barrier. Recent efforts have focused on the development of novel drug delivery methods to enhance brain penetration, but few efforts have aimed at improving its half-life. To explore the behavioral efficacy of an OT analog with enhanced plasma stability, we developed PF-06655075 (PF1), a novel non-brain-penetrant OT receptor agonist with increased selectivity for the OT receptor and significantly increased pharmacokinetic stability. PF-06478939 was generated with only increased stability to disambiguate changes to selectivity versus stability. The efficacy of these compounds in evoking behavioral effects was tested in a conditioned fear paradigm. Both central and peripheral administration of PF1 inhibited freezing in response to a conditioned fear stimulus. Peripheral administration of PF1 resulted in a sustained level of plasma concentrations for greater than 20 hours but no detectable accumulation in brain tissue, suggesting that plasma or cerebrospinal fluid exposure was sufficient to evoke behavioral effects. Behavioral efficacy of peripherally administered OT receptor agonists on conditioned fear response opens the door to potential peripheral mechanisms in other behavioral paradigms, whether they are mediated by direct peripheral activation or feed-forward responses. Compound PF1 is freely available as a tool compound to further explore the role of peripheral OT in behavioral response.

Oxytocin in the nucleus accumbens shell reverses CRFR2-evoked passive stress-coping after partner loss in monogamous male prairie voles.

Loss of a partner can have severe effects on mental health. Here we explore the neural mechanisms underlying increased passive stress-coping, indicative of depressive-like behavior, following the loss of the female partner in the monogamous male prairie vole. We demonstrate that corticotropin-releasing factor receptor 2 (CRFR2) in the nucleus accumbens shell mediates social loss-induced passive coping. Further, we show that partner loss compromises the oxytocin system through multiple mechanisms. Finally, we provide evidence for an interaction of the CRFR2 and oxytocin systems in mediating the emotional consequences of partner loss. Our results suggest that chronic activation of CRFR2 and suppression of striatal oxytocin signaling following partner loss result in an aversive emotional state that may share underlying mechanisms with bereavement. We propose that the suppression of oxytocin signaling is likely adaptive during short separations to encourage reunion with the partner and may have evolved to maintain long-term partnerships. Additionally, therapeutic strategies targeting these systems should be considered for treatment of social loss-mediated depression.

Melanocortin Receptor Agonists Facilitate Oxytocin-Dependent Partner Preference Formation in the Prairie Vole.

The central melanocortin (MC) system has been widely studied for its effects on food intake and sexual behavior. However, the MC system, and more specifically the MC4 receptor (MC4R), also interacts with neurochemical systems that regulate socioemotional behaviors, including oxytocin (OT) and dopamine. In monogamous prairie voles, OT and dopamine interact to promote partner preference formation, a laboratory measure of an enduring social bond between mates. Here we investigated the effects of MC receptor activation on partner preference formation in prairie voles, as well as the interaction between the MC and OT systems during this process. Peripheral administration of the brain penetrant MC3/4R receptor peptide agonist, Melanotan II (MTII), and the highly selective, small-molecule MC4R agonist, Pf-446687, enhanced partner preference formation in the prairie vole, but not in the non-monogamous meadow vole. MTII-induced partner preferences were enduring, as they were present 1 week after drug manipulation. The prosocial effects of MCR agonists may be mediated, in part, through modulation of OT, as coadministration of an OT receptor antagonist prevented MTII-induced partner preferences. MTII also selectively activated hypothalamic OT neurons and potentiated central OT release. As OT has been shown to enhance some aspects of social cognition in humans, our data suggest that the MC4R may be a viable therapeutic target for enhancing social function in psychiatric disorders, including autism spectrum disorders and schizophrenia, potentially through activation of the OT system.

Neonatal melanocortin receptor agonist treatment reduces play fighting and promotes adult attachment in prairie voles in a sex-dependent manner.

The melanocortin receptor (MCR) system has been studied extensively for its role in feeding and sexual behavior, but effects on social behavior have received little attention. α-MSH interacts with neural systems involved in sociality, including oxytocin, dopamine, and opioid systems. Acute melanotan-II (MTII), an MC3/4R agonist, potentiates brain oxytocin (OT) release and facilitates OT-dependent partner preference formation in socially monogamous prairie voles. Here we examined the long-term impact of early-life MCR stimulation on hypothalamic neuronal activity and social development in prairie voles. Male and female voles were given daily subcutaneous injections of 10 mg/kg MTII or saline between postnatal days (PND) 1-7. Neonatally-treated males displayed a reduction in initiated play fighting bouts as juveniles compared to control males. Neonatal exposure to MTII facilitated partner preference formation in adult females, but not males, after a brief cohabitation with an opposite-sex partner. Acute MTII injection elicited a significant burst of the immediate early gene EGR-1 immunoreactivity in hypothalamic OT, vasopressin, and corticotrophin releasing factor neurons, when tested in PND 6-7 animals. Daily neonatal treatment with 1 mg/kg of a more selective, brain penetrant MC4R agonist, PF44687, promoted adult partner preferences in both females and males compared with vehicle controls. Thus, developmental exposure to MCR agonists lead to a persistent change in social behavior, suggestive of structural or functional changes in the neural circuits involved in the formation of social relationships.

Aerosolized oxytocin increases cerebrospinal fluid oxytocin in rhesus macaques.

Intranasal (IN) administration is a widely used method for examining the effect of oxytocin (OT) on social behavior and cognition in healthy subjects and psychiatric populations. IN-OT in humans enhances trust, emotional perception, and empathetic behavior and is under investigation as a potential pharmacotherapy to enhance social functioning in a variety of neuropsychiatric disorders, including autism spectrum disorders (ASD). Nonhuman primates (NHP) are an important model for understanding the effect of OT on social cognition, its neural mechanisms, and the development of IN-OT as a pharmacotherapy for treating social deficits in humans. However, NHP and even some human populations, such as very young infants and children, cannot easily follow the detailed self-administration protocol used in the majority of human IN-OT studies. Therefore, we evaluated the efficacy of several OT-administration routes for elevating central OT concentrations in rhesus macaques. First, we examined the effect of IN and intravenous (IV) routes of OT administration on concentrations of OT and vasopressin (AVP) in plasma and lumbar CSF. Second, we examined these same measures in monkeys after an aerosolized (AE) OT delivery route. All three administration routes significantly increased plasma OT concentrations, but only the AE-OT route significantly increased concentrations of CSF OT. No route affected concentrations of AVP in plasma or CSF. This study confirms that the AE route is the most effective method for increasing central OT concentrations in monkeys, and may also be an effective route, alternative to IN, for administering OT to some human populations.