Implementation and Evaluation of a Bystander Naloxone Training Course.

Introduction: Bystander provision of naloxone is a key modality to reduce opioid overdose-related death. Naloxone training courses are available, but no standardized program exists. As part of a bystander empowerment course, we created and evaluated a brief naloxone training module. Methods: This was a retrospective evaluation of a naloxone training course, which was paired with Stop the Bleed training for hemorrhage control and was offered to administrative staff in an office building. Participants worked in an organization related to healthcare, but none were clinicians. The curriculum included the following topics: 1) background about the opioid epidemic; 2) how to recognize the signs of an opioid overdose; 3) actions not to take when encountering an overdose victim; 4) the correct steps to take when encountering an overdose victim; 5) an overview of naloxone products; and 6) Good Samaritan protection laws. The 20-minute didactic section was followed by a hands-on session with nasal naloxone kits and a simulation mannequin. The course was evaluated with the Opioid Overdose Knowledge (OOKS) and Opioid Overdose Attitudes (OOAS) scales for take-home naloxone training evaluation. We used the paired Wilcoxon signed-rank test to compare scores pre- and post-course. Results: Twenty-eight participants completed the course. The OOKS, measuring objective knowledge about opioid overdose and naloxone, had improved scores from a median of 73.2% (interquartile range [IQR] 68.3%-79.9%) to 91.5% (IQR 85.4%-95.1%), P < 0.001. The three domains on the OOAS score also showed statistically significant results. Competency to manage an overdose improved on a five-point scale from a median of 2.5 (IQR 2.4-2.9) to a median of 3.7 (IQR 3.5-4.1), P < 0.001. Concerns about managing an overdose decreased (improved) from a median of 2.3 (IQR 1.9-2.6) to median 1.8 (IQR 1.5-2.1), P < 0.001. Readiness to intervene in an opioid overdose improved from a median of 4 (IQR 3.8-4.2) to a median of 4.2 (IQR 4-4.2), P < 0.001. Conclusion: A brief course designed to teach bystanders about opioid overdose and naloxone was feasible and effective. We encourage hospitals and other organizations to use and promulgate this model. Furthermore, we suggest the convening of a national consortium to achieve consensus on program content and delivery.

Active vision in freely moving marmosets using head-mounted eye tracking.

Our understanding of how vision functions as primates actively navigate the real-world is remarkably sparse. As most data have been limited to chaired and typically head-restrained animals, the synergistic interactions of different motor actions/plans inherent to active sensing - e.g. eyes, head, posture, movement, etc. - on visual perception are largely unknown. To address this considerable gap in knowledge, we developed an innovative wireless head-mounted eye tracking system called CEREBRO for small mammals, such as marmoset monkeys. Our system performs Chair-free Eye-Recording using Backpack mounted micROcontrollers. Because eye illumination and environment lighting change continuously in natural contexts, we developed a segmentation artificial neural network to perform robust pupil tracking in these conditions. Leveraging this innovative system to investigate active vision, we demonstrate that although freely-moving marmosets exhibit frequent compensatory eye movements equivalent to other primates, including humans, the predictability of the visual system is enhanced when animals are freely-moving relative to when they are head-fixed. Moreover, despite increases in eye/head-motion during locomotion, gaze stabilization actually improved over periods when the monkeys were stationary. Rather than impair vision, the dynamics of gaze stabilization in freely-moving primates has been optimized over evolution to enable active sensing during natural exploration.

An ethologically motivated neurobiology of primate visually-guided reach-to-grasp behavior.

The precision of primate visually guided reaching likely evolved to meet the many challenges faced by living in arboreal environments, yet much of what we know about the underlying primate brain organization derives from a set of highly constrained experimental paradigms. Here we review the role of vision to guide natural reach-to-grasp movements in marmoset monkey prey capture to illustrate the breadth and diversity of these behaviors in ethological contexts, the fast predictive nature of these movements [1,2], and the advantages of this particular primate model to investigate the underlying neural mechanisms in more naturalistic contexts [3]. In addition to their amenability to freely-moving neural recording methods for investigating the neural basis of dynamic ethological behaviors [4,5], marmosets have a smooth neocortical surface that facilitates imaging and array recordings [6,7] in all areas in the primate fronto-parietal network [8,9]. Together, this model organism offers novel opportunities to study the real-world interplay between primate vision and reach-to-grasp dynamics using ethologically motivated neuroscientific experimental designs.

Long-range projections of oxytocin neurons in the marmoset brain.

The neurohormone oxytocin (OT) has become a major target for the development of novel therapeutic strategies to treat psychiatric disorders such as autism spectrum disorder because of its integral role in governing many facets of mammalian social behavior. Whereas extensive work in rodents has produced much of our knowledge of OT, we lack basic information about its neurobiology in primates making it difficult to interpret the limited effects that OT manipulations have had in human patients. In fact, previous studies have revealed only limited OT fibers in primate brains. Here, we investigated the OT connectome in marmoset using immunohistochemistry, and mapped OT fibers throughout the brains of adult male and female marmoset monkeys. We found extensive OT projections reaching limbic and cortical areas that are involved in the regulation of social behaviors, such as the amygdala, the medial prefrontal cortex, and the basal ganglia. The pattern of OT fibers observed in marmosets is notably similar to the OT connectomes described in rodents. Our findings here contrast with previous results by demonstrating a broad distribution of OT throughout the marmoset brain. Given the prevalence of this neurohormone in the primate brain, methods developed in rodents to manipulate endogenous OT are likely to be applicable in marmosets.

Representing the dynamics of natural marmoset vocal behaviors in frontal cortex.

Here we tested the respective contributions of primate premotor and prefrontal cortex to support vocal behavior. We applied a model-based GLM analysis that better accounts for the inherent variance in natural, continuous behaviors to characterize the activity of neurons throughout frontal cortex as freely-moving marmosets engaged in conversational exchanges. While analyses revealed functional clusters of neural activity related to the different processes involved in the vocal behavior, these clusters did not map to subfields of prefrontal or premotor cortex, as has been observed in more conventional task-based paradigms. Our results suggest a distributed functional organization for the myriad neural mechanisms underlying natural social interactions and has implications for our concepts of the role that frontal cortex plays in governing ethological behaviors in primates.

Long range projections of oxytocin neurons in the marmoset brain.

The neurohormone oxytocin (OT) has become a major target for the development of novel therapeutic strategies to treat psychiatric disorders such as autism spectrum disorder because of its integral role in governing many facets of mammalian social behavior. Whereas extensive work in rodents has produced much of our knowledge of OT, we lack basic information about its neurobiology in primates making it difficult to interpret the limited effects that OT manipulations have had in human patients. In fact, previous studies have revealed only limited OT fibers in primate brains. Here, we investigated the OT connectome in marmoset using immunohistochemistry, and mapped OT fibers throughout the brains of adult male and female marmoset monkeys. We found extensive OT projections reaching limbic and cortical areas that are involved in the regulation of social behaviors, such as the amygdala, the medial prefrontal cortex and the basal ganglia. The pattern of OT fibers observed in marmosets is notably similar to the OT connectomes described in rodents. Our findings here contrast with previous results by demonstrating a broad distribution of OT throughout the marmoset brain. Given the prevalence of this neurohormone in the primate brain, methods developed in rodents to manipulate endogenous OT are likely to be applicable in marmosets.

Cross-modal representation of identity in the primate hippocampus.

Faces and voices are the dominant social signals used to recognize individuals among primates. Yet, it is not known how these signals are integrated into a cross-modal representation of individual identity in the primate brain. We discovered that, although single neurons in the marmoset hippocampus exhibited selective responses when presented with the face or voice of a specific individual, a parallel mechanism for representing the cross-modal identities for multiple individuals was evident within single neurons and at the population level. Manifold projections likewise showed the separability of individuals as well as clustering for others' families, which suggests that multiple learned social categories are encoded as related dimensions of identity in the hippocampus. Neural representations of identity in the hippocampus are thus both modality independent and reflect the primate social network.

The neurobiology of vocal communication in marmosets.

An increasingly popular animal model for studying the neural basis of social behavior, cognition, and communication is the common marmoset (Callithrix jacchus). Interest in this New World primate across neuroscience is now being driven by their proclivity for prosociality across their repertoire, high volubility, and rapid development, as well as their amenability to naturalistic testing paradigms and freely moving neural recording and imaging technologies. The complement of these characteristics set marmosets up to be a powerful model of the primate social brain in the years to come. Here, we focus on vocal communication because it is the area that has both made the most progress and illustrates the prodigious potential of this species. We review the current state of the field with a focus on the various brain areas and networks involved in vocal perception and production, comparing the findings from marmosets to other animals, including humans.

Adeno-associated viral vectors for functional intravenous gene transfer throughout the non-human primate brain.

Crossing the blood-brain barrier in primates is a major obstacle for gene delivery to the brain. Adeno-associated viruses (AAVs) promise robust, non-invasive gene delivery from the bloodstream to the brain. However, unlike in rodents, few neurotropic AAVs efficiently cross the blood-brain barrier in non-human primates. Here we report on AAV.CAP-Mac, an engineered variant identified by screening in adult marmosets and newborn macaques, which has improved delivery efficiency in the brains of multiple non-human primate species: marmoset, rhesus macaque and green monkey. CAP-Mac is neuron biased in infant Old World primates, exhibits broad tropism in adult rhesus macaques and is vasculature biased in adult marmosets. We demonstrate applications of a single, intravenous dose of CAP-Mac to deliver functional GCaMP for ex vivo calcium imaging across multiple brain areas, or a cocktail of fluorescent reporters for Brainbow-like labelling throughout the macaque brain, circumventing the need for germline manipulations in Old World primates. As such, CAP-Mac is shown to have potential for non-invasive systemic gene transfer in the brains of non-human primates.

Functional gene delivery to and across brain vasculature of systemic AAVs with endothelial-specific tropism in rodents and broad tropism in primates.

Delivering genes to and across the brain vasculature efficiently and specifically across species remains a critical challenge for addressing neurological diseases. We have evolved adeno-associated virus (AAV9) capsids into vectors that transduce brain endothelial cells specifically and efficiently following systemic administration in wild-type mice with diverse genetic backgrounds, and in rats. These AAVs also exhibit superior transduction of the CNS across non-human primates (marmosets and rhesus macaques), and in ex vivo human brain slices, although the endothelial tropism is not conserved across species. The capsid modifications translate from AAV9 to other serotypes such as AAV1 and AAV-DJ, enabling serotype switching for sequential AAV administration in mice. We demonstrate that the endothelial-specific mouse capsids can be used to genetically engineer the blood-brain barrier by transforming the mouse brain vasculature into a functional biofactory. We apply this approach to Hevin knockout mice, where AAV-X1-mediated ectopic expression of the synaptogenic protein Sparcl1/Hevin in brain endothelial cells rescued synaptic deficits.

Intravenous functional gene transfer throughout the brain of non-human primates using AAV.

Adeno-associated viruses (AAVs) promise robust gene delivery to the brain through non-invasive, intravenous delivery. However, unlike in rodents, few neurotropic AAVs efficiently cross the blood-brain barrier in non-human primates (NHPs). Here we describe AAV.CAP-Mac, an engineered variant identified by screening in adult marmosets and newborn macaques with improved efficiency in the brain of multiple NHP species: marmoset, rhesus macaque, and green monkey. CAP-Mac is neuron-biased in infant Old World primates, exhibits broad tropism in adult rhesus macaques, and is vasculature-biased in adult marmosets. We demonstrate applications of a single, intravenous dose of CAP-Mac to deliver (1) functional GCaMP for ex vivo calcium imaging across multiple brain areas, and (2) a cocktail of fluorescent reporters for Brainbow-like labeling throughout the macaque brain, circumventing the need for germline manipulations in Old World primates. Given its capabilities for systemic gene transfer in NHPs, CAP-Mac promises to help unlock non-invasive access to the brain.

Functional gene delivery to and across brain vasculature of systemic AAVs with endothelial-specific tropism in rodents and broad tropism in primates.

Delivering genes to and across the brain vasculature efficiently and specifically across species remains a critical challenge for addressing neurological diseases. We have evolved adeno-associated virus (AAV9) capsids into vectors that transduce brain endothelial cells specifically and efficiently following systemic administration in wild-type mice with diverse genetic backgrounds and rats. These AAVs also exhibit superior transduction of the CNS across non-human primates (marmosets and rhesus macaques), and ex vivo human brain slices although the endothelial tropism is not conserved across species. The capsid modifications translate from AAV9 to other serotypes such as AAV1 and AAV-DJ, enabling serotype switching for sequential AAV administration in mice. We demonstrate that the endothelial specific mouse capsids can be used to genetically engineer the blood-brain barrier by transforming the mouse brain vasculature into a functional biofactory. Vasculature-secreted Hevin (a synaptogenic protein) rescued synaptic deficits in a mouse model.

Prevalence and Risk Factors of Major Depression in Patients with Diabetic Retinopathy in a Nationally Representative Survey.

PURPOSE: Evaluate the prevalence and risk factors of depression in diabetic retinopathy (DR). Compare subjective and objective measures of visual function predictivity of depression. METHODS: National Health and Nutrition Examination Survey 2005-2008 participants aged ≥40 who underwent fundus photography, Patient Health Questionnaire (PHQ)-9, and Visual Function Questionnaire (VFQ-25) were included in the study. Multivariable logistic regression was used to evaluate whether DR was a significant risk factor for depression and to evaluate the risk factors for depression in those with DR. RESULTS: A total of 5704 participants, 47% male, and mean age 56.5 years were included in this study. Persons with moderate, severe non-proliferative diabetic retinopathy (NPDR), or proliferative retinopathy (PDR) had higher prevalence of depression than participants with mild retinopathy or no retinopathy (14.3%, 6.9%, 7.0%). Moderate-to-severe NPDR or PDR (OR: 2.36, p = .04) was associated with depression. Among persons with DR, best-corrected visual acuity and HbA1c were not associated with depression. However, self-reported measures of vision were associated with depression: some of the time spent worrying about eyesight (OR: 4.59, p = .010), vision limit activities some of the time (OR: 8.52, p < .001), vision limits activities most/all of the time (OR: 6.99, p < .001). CONCLUSIONS: A significant proportion of patients with DR in the NHANES population had co-morbid major depression. Best corrected visual acuity was not associated with depression in those with DR, while subjective, self-reported measures were associated with depression, suggesting subjective measures are a better determinant of poor mood and low functional status.

Engineered AAVs for non-invasive gene delivery to rodent and non-human primate nervous systems.

Gene therapy offers great promise in addressing neuropathologies associated with the central and peripheral nervous systems (CNS and PNS). However, genetic access remains difficult, reflecting the critical need for the development of effective and non-invasive gene delivery vectors across species. To that end, we evolved adeno-associated virus serotype 9 (AAV9) capsid in mice and validated two capsids, AAV-MaCPNS1 and AAV-MaCPNS2, across rodent species (mice and rats) and non-human primate (NHP) species (marmosets and rhesus macaques). Intravenous administration of either AAV efficiently transduced the PNS in rodents and both the PNS and CNS in NHPs. Furthermore, we used AAV-MaCPNS1 in mice to systemically deliver the following: (1) the neuronal sensor jGCaMP8s to record calcium signal dynamics in nodose ganglia and (2) the neuronal actuator DREADD to dorsal root ganglia to mediate pain. This conclusively demonstrates the translatability of these two systemic AAVs across four species and their functional utility through proof-of-concept studies in mice.

Active vision during prey capture in wild marmoset monkeys.

A foundational pressure in the evolution of all animals is the ability to travel through the world, inherently coupling the sensory and motor systems. While this relationship has been explored in several species,1-4 it has been largely overlooked in primates, which have typically relied on paradigms in which head-restrained subjects view stimuli on screens.5 Natural visual behaviors, by contrast, are typified by locomotion through the environment guided by active sensing as animals explore and interact with the world,4,6 a relationship well illustrated by prey capture.7-12 Here, we characterized prey capture in wild marmoset monkeys as they negotiated their dynamic, arboreal habitat to illustrate the inherent role of vision as an active process in natural nonhuman primate behavior. Not only do marmosets share the core properties of vision that typify the primate Order,13-18 but they are prolific hunters that prey on a diverse set of prey animals.19-22 Marmosets pursued prey using vision in several different contexts, but executed precise visually guided motor control that predominantly involved grasping with hands for successful capture of prey. Applying markerless tracking for the first time in wild primates yielded novel findings that precisely quantified how marmosets track insects prior to initiating an attack and the rapid visually guided corrections of the hands during capture. These findings offer the first detailed insight into the active nature of vision to guide multiple facets of a natural goal-directed behavior in wild primates and can inform future laboratory studies of natural primate visual behaviors and the supporting neural processes.

Natural behavior is the language of the brain.

The breadth and complexity of natural behaviors inspires awe. Understanding how our perceptions, actions, and internal thoughts arise from evolved circuits in the brain has motivated neuroscientists for generations. Researchers have traditionally approached this question by focusing on stereotyped behaviors, either natural or trained, in a limited number of model species. This approach has allowed for the isolation and systematic study of specific brain operations, which has greatly advanced our understanding of the circuits involved. At the same time, the emphasis on experimental reductionism has left most aspects of the natural behaviors that have shaped the evolution of the brain largely unexplored. However, emerging technologies and analytical tools make it possible to comprehensively link natural behaviors to neural activity across a broad range of ethological contexts and timescales, heralding new modes of neuroscience focused on natural behaviors. Here we describe a three-part roadmap that aims to leverage the wealth of behaviors in their naturally occurring distributions, linking their variance with that of underlying neural processes to understand how the brain is able to successfully navigate the everyday challenges of animals' social and ecological landscapes. To achieve this aim, experimenters must harness one challenge faced by all neurobiological systems, namely variability, in order to gain new insights into the language of the brain.

Behavioral context affects social signal representations within single primate prefrontal cortex neurons.

We tested whether social signal processing in more traditional, head-restrained contexts is representative of the putative natural analog-social communication-by comparing responses to vocalizations within individual neurons in marmoset prefrontal cortex (PFC) across a series of behavioral contexts ranging from traditional to naturalistic. Although vocalization-responsive neurons were evident in all contexts, cross-context consistency was notably limited. A response to these social signals when subjects were head-restrained was not predictive of a comparable neural response to the identical vocalizations during natural communication. This pattern was evident both within individual neurons and at a population level, as PFC activity could be reliably decoded for the behavioral context in which vocalizations were heard. These results suggest that neural representations of social signals in primate PFC are not static but highly flexible and likely reflect how nuances of the dynamic behavioral contexts affect the perception of these signals and what they communicate.

Continuous Renal Replacement Therapy for Two Neonates With Hyperammonemia.

Objectives: This study aims to assess the feasibility of using hemofiltration for ammonia clearance in low body weight infants with an inborn error of metabolism. Design: A study of two cases. Setting: Quaternary pediatric hospital (Saint Louis Children's Hospital) NICU and PICU. Patients: Infants <6 months of age with an ICD-9 diagnosis of 270.6 (hyperammonemia). Interventions: Continuous renal replacement therapy (CRRT). Measurements and Main Results: We measure serum ammonia levels over time and the rate of ammonia clearance over time. Continuous renal replacement therapy was more effective than scavenger therapy alone (Ammonul™) for rapid removal of ammonia in low weight infants (as low as 2.5 kg). Conclusions: Continuous renal replacement therapy is technically feasible in low weight infants with severe hyperammonemia secondary to an inborn error of metabolism.

Current practices in nutrition management and disease incidence of common marmosets (Callithrix jacchus).

BACKGROUND: A survey was developed to characterize disease incidence, common pathology lesions, environmental characteristics, and nutrition programs within captive research marmoset colonies. METHODS: Seventeen research facilities completed the electronic survey. RESULTS: Nutritional management programs varied amongst research institutions housing marmosets; eight primary base diets were reported. The most common clinical syndromes reported were gastrointestinal disease (i.e. inflammatory bowel disease like disease, chronic lymphocytic enteritis, chronic malabsorption, chronic diarrhea), metabolic bone disease or fracture, infectious diarrhea, and oral disease (tooth root abscesses, gingivitis, tooth root resorption). The five most common pathology morphologic diagnoses were colitis, nephropathy/nephritis, enteritis, chronic lymphoplasmacytic enteritis, and cholecystitis. Obesity was more common (average 20% of a reporting institution's population) than thin body condition (average 5%). CONCLUSIONS: Through review of current practices, we aim to inspire development of evidence-based practices to standardize husbandry and nutrition practices for marmoset research colonies.