Modelling behaviors relevant to brain disorders in the nonhuman primate: Are we there yet?

Recent years have seen a profound resurgence of activity with nonhuman primates (NHPs) to model human brain disorders. From marmosets to macaques, the study of NHP species offers a unique window into the function of primate-specific neural circuits that are impossible to examine in other models. Examining how these circuits manifest into the complex behaviors of primates, such as advanced cognitive and social functions, has provided enormous insights to date into the mechanisms underlying symptoms of numerous neurological and neuropsychiatric illnesses. With the recent optimization of modern techniques to manipulate and measure neural activity in vivo, such as optogenetics and calcium imaging, NHP research is more well-equipped than ever to probe the neural mechanisms underlying pathological behavior. However, methods for behavioral experimentation and analysis in NHPs have noticeably failed to keep pace with these advances. As behavior ultimately lies at the junction between preclinical findings and its translation to clinical outcomes for brain disorders, approaches to improve the integrity, reproducibility, and translatability of behavioral experiments in NHPs requires critical evaluation. In this review, we provide a unifying account of existing brain disorder models using NHPs, and provide insights into the present and emerging contributions of behavioral studies to the field.

NogoA-expressing astrocytes limit peripheral macrophage infiltration after ischemic brain injury in primates.

Astrocytes play critical roles after brain injury, but their precise function is poorly defined. Utilizing single-nuclei transcriptomics to characterize astrocytes after ischemic stroke in the visual cortex of the marmoset monkey, we observed nearly complete segregation between stroke and control astrocyte clusters. Screening for the top 30 differentially expressed genes that might limit stroke recovery, we discovered that a majority of astrocytes expressed RTN4A/ NogoA, a neurite-outgrowth inhibitory protein previously only associated with oligodendrocytes. NogoA upregulation on reactive astrocytes post-stroke was significant in both the marmoset and human brain, whereas only a marginal change was observed in mice. We determined that NogoA mediated an anti-inflammatory response which likely contributes to limiting the infiltration of peripheral macrophages into the surviving parenchyma.

Visual Cortical Area MT Is Required for Development of the Dorsal Stream and Associated Visuomotor Behaviors.

The middle temporal (MT) area of the extrastriate visual cortex has long been studied in adulthood for its distinctive physiological properties and function as a part of the dorsal stream, yet interestingly it possesses a similar maturation profile as the primary visual cortex (V1). Here, we examined whether an early-life lesion in MT of marmoset monkeys (six female, two male) altered the dorsal stream development and the behavioral precision of reaching-to-grasp sequences. We observed permanent changes in the anatomy of cortices associated with both reaching (parietal and medial intraparietal areas) and grasping (anterior intraparietal area), as well as in reaching-and-grasping behaviors. In addition, we observed a significant impact on the anatomy of V1 and the direction sensitivity of V1 neurons in the lesion projection zone. These findings indicate that area MT is a crucial node in the development of primate vision, affecting both V1 and areas in the dorsal visual pathway known to mediate visually guided manual behaviors.SIGNIFICANCE STATEMENT Previous studies have identified a role for the MT area of the visual cortex in perceiving motion, yet none have examined its central role in the development of the visual cortex and in the establishment of visuomotor behaviors. To address this, we used a unilateral MT lesion model in neonatal marmosets before examining the anatomic, physiological, and behavioral consequences. In adulthood, we observed perturbations in goal-orientated reach-and-grasp behavior, altered direction selectivity of V1 neurons, and changes in the cytoarchitecture throughout dorsal stream areas. This study highlights the importance of MT as a central node in visual system development and consequential visuomotor activity.

Replicating infant-specific reactive astrocyte functions in the injured adult brain.

Infants and adults respond differently to brain injuries. Specifically, improved neuronal sparing along with reduced astrogliosis and glial scarring often observed earlier in life, likely contributes to improved long-term outcomes. Understanding the underlying mechanisms could enable the recapitulation of neuroprotective effects, observed in infants, to benefit adults after brain injuries. We reveal that in primates, Eph/ ephrin signaling contributes to age-dependent reactive astrocyte behavior. Ephrin-A5 expression on astrocytes was more protracted in adults, whereas ephrin-A1 was only expressed on infant astrocytes. Furthermore, ephrin-A5 exacerbated major hallmarks of astrocyte reactivity via EphA2 and EphA4 receptors, which was subsequently alleviated by ephrin-A1. Rather than suppressing reactivity, ephrin-A1 signaling shifted astrocytes towards GAP43+ neuroprotection, accounting for improved neuronal sparing in infants. Reintroducing ephrin-A1 after middle-aged focal ischemic injury significantly attenuated glial scarring, improved neuronal sparing and preserved circuitry. Therefore, beneficial infant mechanisms can be recapitulated in adults to improve outcomes after CNS injuries.

Retinal ganglion cells projecting to superior colliculus and pulvinar in marmoset.

We determined the retinal ganglion cell types projecting to the medial subdivision of inferior pulvinar (PIm) and the superior colliculus (SC) in the common marmoset monkey, Callithrix jacchus. Adult marmosets received a bidirectional tracer cocktail into the PIm (conjugated to Alexa fluor 488), and the SC (conjugated to Alexa fluor 594) using an MRI-guided approach. One SC injection included the pretectum. The large majority of retrogradely labelled cells were obtained from SC injections, with only a small proportion obtained after PIm injections. Retrogradely labelled cells were injected intracellularly in vitro using lipophilic dyes (DiI, DiO). The SC and PIm both received input from a variety of ganglion cell types. Input to the PIm was dominated by broad thorny (41%), narrow thorny (24%) and large bistratified (25%) ganglion cells. Input to the SC was dominated by parasol (37%), broad thorny (24%) and narrow thorny (17%) cells. Midget ganglion cells (which make up the large majority of primate retinal ganglion cells) and small bistratified (blue-ON/yellow OFF) cells were never observed to project to SC or PIm. Small numbers of other wide-field ganglion cell types were also encountered. Giant sparse (presumed melanopsin-expressing) cells were only seen following the tracer injection which included the pretectum. We note that despite the location of pulvinar complex in dorsal thalamus, and its increased size and functional importance in primate evolution, the retinal projections to pulvinar have more in common with SC projections than they do with projections to the dorsal lateral geniculate nucleus.

Mapping the neural circuitry of predator fear in the nonhuman primate.

In rodents, innate and learned fear of predators depends on the medial hypothalamic defensive system, a conserved brain network that lies downstream of the amygdala and promotes avoidance via projections to the periaqueductal gray. Whether this network is involved in primate fear remains unknown. To address this, we provoked flight responses to a predator (moving snake) in the marmoset monkey under laboratory conditions. We combined c-Fos immunolabeling and anterograde/retrograde tracing to map the functional connectivity of the ventromedial hypothalamus, a core node in the medial hypothalamic defensive system. Our findings demonstrate that the ventromedial hypothalamus is recruited by predator exposure in primates and that anatomical connectivity of the rodent and primate medial hypothalamic defensive system are highly conserved.

The Age-Dependent Neural Substrates of Blindsight.

Some patients who are considered cortically blind due to the loss of their primary visual cortex (V1) show a remarkable ability to act upon or discriminate between visual stimuli presented to their blind field, without any awareness of those stimuli. This phenomenon is often referred to as blindsight. Despite the range of spared visual abilities, the identification of the pathways mediating blindsight remains an active and contentious topic in the field. In this review, we discuss recent findings of the candidate pathways and their relative contributions to different forms of blindsight across the lifespan to illustrate the varied nature of unconscious visual processing.

The Marmoset: The Next Frontier in Understanding the Development of the Human Brain.

Rodent models, particularly mice, have dominated the field of developmental neuroscience for decades, like they have in most fields of biomedicine research. However, with 80 million years since rodents and primates last shared a common ancestor, the use of mice to model the development of the human brain is not without many shortcomings. The human brain diverges from the mouse brain in many aspects and is comprised of novel structures as well as diversified cellular subtypes. While these newly evolved features have no equivalent in rodents, they are observed in nonhuman primates. Therefore, elucidating the cellular mechanisms underlying the development and maturation of the healthy and diseased human brain can be achieved using less complex nonhuman primates. Historically, macaques were the preferred nonhuman primate model. However, over the past decade, the New World marmoset monkey (Callithrix jacchus) has gained more importance, particularly in the field of neurodevelopment. With its small size, twin or triplet birth, and prosocial behavior, the marmoset is an ideal model to study normal brain development as well as neurodevelopmental disorders, which are often associated with abnormal social behaviors. The growing interest in the marmoset has prompted many comparative studies, all demonstrating that the marmoset brain closely resembles that of the human and is perfectly suited to model human brain development. The marmoset is thus poised to extend its influence in the field of neurodevelopment and will hopefully fill the gaps that the mouse has left in our understanding of how our brain forms and how neurodevelopmental disorders originate.

Extensive Connectivity Between the Medial Pulvinar and the Cortex Revealed in the Marmoset Monkey.

The medial pulvinar (PM) is a multimodal associative thalamic nucleus, recently evolved in primates. PM participates in integrative and modulatory functions, including directed attention, and consistently exhibits alterations in disorders such as schizophrenia and autism. Despite essential cognitive functions, the cortical inputs to the PM have not been systematically investigated. To date, less than 20 cortices have been demonstrated to project to PM. The goal of this study was to establish a comprehensive map of the cortical afferents to PM in the marmoset monkey. Using a magnetic resonance imaging-guided injection approach, we reveal 62 discrete cortices projecting to the adult marmoset PM. We confirmed previously reported connections and identified further projections from discrete cortices across the temporal, parietal, retrosplenial-cingulate, prefrontal, and orbital lobes. These regions encompass areas recipient of PM efferents, demonstrating the reciprocity of the PM-cortical connectivity. Moreover, our results indicate that PM neurones projecting to distinct cortices are intermingled and form multimodal cell clusters. This microunit organization, believed to facilitate cross-modal integration, contrasts with the large functional subdivisions usually observed in thalamic nuclei. Altogether, we provide the first comprehensive map of PM cortical afferents, an essential stepping stone in expanding our knowledge of PM and its function.

Retinotopic specializations of cortical and thalamic inputs to area MT.

Retinotopic specializations in the ventral visual stream, especially foveal adaptations, provide primates with high-acuity vision in the central visual field. However, visual field specializations have not been studied in the dorsal visual stream, dedicated to processing visual motion and visually guided behaviors. To investigate this, we injected retrograde neuronal tracers occupying the whole visuotopic representation of the middle temporal (MT) visual area in marmoset monkeys and studied the distribution and morphology of the afferent primary visual cortex (V1) projections. Contrary to previous reports, we found a heterogeneous population of V1-MT projecting neurons distributed in layers 3C and 6. In layer 3C, spiny stellate neurons were distributed mainly in foveal representations, while pyramidal morphologies were characteristic of peripheral eccentricities. This primate adaptation of the V1 to MT pathway is arranged in a way that we had not previously understood, with abundant stellate projection neurons in the high-resolution foveal portions, suggesting rapid relay of motion information to visual area MT. We also describe that the medial portion of the inferior pulvinar (PIm), which is the main thalamic input to area MT, shows a retinotopic organization, likely reflecting the importance of this pathway during development and the establishment of area MT topography.

Prehensile kinematics of the marmoset monkey: Implications for the evolution of visually-guided behaviors.

Throughout the primate lineage, there is a wide diversity of prehensile capacity that is thought to stem from individual species foraging patterns. While many studies have explored primates with precise hand grips, such as higher apes, few have considered primates that lack opposition movements. The New World marmoset monkey occupies an intriguing niche, displaying adept control of their hand movements yet their absence of opposable digits results in relatively imprecise grasping actions when compared with those observed in Old World monkeys, apes, and humans. The marmoset monkey offers a unique composition of ancestral primate corticospinal organization combined with skilled hand use to explore the evolution and development of visually-guided actions. In this study, four adult marmosets were trained to perform a series of visually-guided tasks, designed to assess their control over locating and retrieving objects of differing dimensions. Two of these animals received a neonatal lesion of the inferior pulvinar (unilateral), a thalamic nucleus previously demonstrated to be involved in visuomotor development. The kinematics of their reaching and grasping patterns were recorded for offline analysis. Predictive modeling revealed that maximum grip aperture, time to reach peak velocity and hand use were reliable predictors of distinguishing between cohorts. A consistent feature observed across all tasks was that they do not precisely scale their grip according to the dimensions of the target object which may be attributed to their lack of independent digit control. Therefore, the marmoset monkey represents a previously understudied position in the evolution of primate reach and grasp behavior.

The medial pulvinar: function, origin and association with neurodevelopmental disorders.

The pulvinar is primarily referred to for its role in visual processing. However, the 'visual pulvinar' only encompasses the inferior and lateral regions of this complex thalamic nucleus. The remaining medial portion (medial pulvinar, PM) establishes distinct cortical connectivity and has been associated with directed attention, executive functions and working memory. These functions are particularly impaired in neurodevelopmental disorders, including schizophrenia and attention deficit and hyperactivity disorder (ADHD), both of which have been associated with abnormal PM architecture and connectivity. With these disorders becoming more prevalent in modern societies, we review the literature to better understand how the PM can participate in the pathophysiology of cognitive disorders and how a better understanding of the development and function of this thalamic nucleus, which is most likely exclusive to the primate brain, can advance clinical research and treatments.

Thalamocortical Afferents Innervate the Cortical Subplate much Earlier in Development in Primate than in Rodent.

The current model, based on rodent data, proposes that thalamocortical afferents (TCA) innervate the subplate towards the end of cortical neurogenesis. This implies that the laminar identity of cortical neurons is specified by intrinsic instructions rather than information of thalamic origin. In order to determine whether this mechanism is conserved in the primates, we examined the growth of thalamocortical (TCA) and corticofugal afferents in early human and monkey fetal development. In the human, TCA, identified by secretagogin, calbindin, and ROBO1 immunoreactivity, were observed in the internal capsule of the ventral telencephalon as early as 7-7.5 PCW, crossing the pallial/subpallial boundary (PSB) by 8 PCW before the calretinin immunoreactive corticofugal fibers do. Furthermore, TCA were observed to be passing through the intermediate zone and innervating the presubplate of the dorsolateral cortex, and already by 10-12 PCW TCAs were occupying much of the cortex. Observations at equivalent stages in the marmoset confirmed that this pattern is conserved across primates. Therefore, our results demonstrate that in primates, TCAs innervate the cortical presubplate at earlier stages than previously demonstrated by acetylcholinesterase histochemistry, suggesting that pioneer thalamic afferents may contribute to early cortical circuitry that can participate in defining cortical neuron phenotypes.

Unravelling the subcortical and retinal circuitry of the primate inferior pulvinar.

The primate visual brain possesses a myriad of pathways, whereby visual information originating at the retina is transmitted to multiple subcortical areas in parallel, before being relayed onto the visual cortex. The dominant retinogeniculostriate pathway has been an area of extensive study, and Vivien Casagrande's work in examining the once overlooked koniocellular pathway of the lateral geniculate nucleus has generated interest in how alternate subcortical pathways can contribute to visual perception. Another subcortical visual relay center is the inferior pulvinar (PI), which has four subdivisions and numerous connections with other subcortical and cortical areas and is directly recipient of retinal afferents. The complexity of subcortical connections associated with the PI subdivisions has led to differing results from various groups. A particular challenge in determining the exact connectivity pattern has been in accurately targeting the subdivisions of the PI with neural tracers. Therefore, in the present study, we used a magnetic resonance imaging (MRI)-guided stereotaxic injection system to inject bidirectional tracers in the separate subdivisions of the PI, the superior layers of the superior colliculus, the retina, and the lateral geniculate nucleus. Our results have determined for the first time that the medial inferior pulvinar (PIm) is innervated by widefield retinal ganglion cells (RGCs), and this pathway is not a collateral branch of the geniculate and collicular projecting RGCs. Furthermore, our tracing data shows no evidence of collicular terminations in the PIm, which are confined to the centromedial and posterior PI.

More than blindsight: Case report of a child with extraordinary visual capacity following perinatal bilateral occipital lobe injury.

Injury to the primary visual cortex (V1, striate cortex) and the geniculostriate pathway in adults results in cortical blindness, abolishing conscious visual perception. Early studies by Larry Weiskrantz and colleagues demonstrated that some patients with an occipital-lobe injury exhibited a degree of unconscious vision and visually-guided behaviour within the blind field. A more recent focus has been the observed phenomenon whereby early-life injury to V1 often results in the preservation of visual perception in both monkeys and humans. These findings initiated a concerted effort on multiple fronts, including nonhuman primate studies, to uncover the neural substrate/s of the spared conscious vision. In both adult and early-life cases of V1 injury, evidence suggests the involvement of the Middle Temporal area (MT) of the extrastriate visual cortex, which is an integral component area of the dorsal stream and is also associated with visually-guided behaviors. Because of the limited number of early-life V1 injury cases for humans, the outstanding question in the field is what secondary visual pathways are responsible for this extraordinary capacity? Here we report for the first time a case of a child (B.I.) who suffered a bilateral occipital-lobe injury in the first two weeks postnatally due to medium-chain acyl-Co-A dehydrogenase deficiency. At 6 years of age, B.I. underwent a battery of neurophysiological tests, as well as structural and diffusion MRI and ophthalmic examination at 7 years. Despite the extensive bilateral occipital cortical damage, B.I. has extensive conscious visual abilities, is not blind, and can use vision to navigate his environment. Furthermore, unlike blindsight patients, he can readily and consciously identify happy and neutral faces and colors, tasks associated with ventral stream processing. These findings suggest significant re-routing of visual information. To identify the putative visual pathway/s responsible for this ability, MRI tractography of secondary visual pathways connecting MT with the lateral geniculate nucleus (LGN) and the inferior pulvinar (PI) were analysed. Results revealed an increased PI-MT pathway in the left hemisphere, suggesting that this pulvinar relay could be the neural pathway affording the preserved visual capacity following an early-life lesion of V1. These findings corroborate anatomical evidence from monkeys showing an enhanced PI-MT pathway following an early-life lesion of V1, compared to adults.

Full: Ontogenesis and development of the nonhuman primate pulvinar.

Recent evidence demonstrates that the pulvinar nuclei play a critical role in shaping the connectivity and function of the multiple cortical areas they connect. Surprisingly, however, little is known about the development of this area, the largest corpus of the thalamic nuclei, which go on to occupy 40% of the adult thalamus in the human. It was proposed that the nonhuman primate and the human pulvinar develop according to very different processes, with a greatly reduced neurogenic period in nonhuman primate compared to human and divergent origins. In the marmoset monkey, we demonstrate that neurons populating the pulvinar are generated throughout gestation, suggesting that this aspect of development is more similar to the human than first predicted. While we were able to confirm the diencephalic source of pulvinar neurons, we provide new evidence contesting the presence of an additional niche in the telencephalon. Finally, our study defines new molecular markers that will simplify future investigations in the development and evolution of the pulvinar.

Acute or Delayed Systemic Administration of Human Amnion Epithelial Cells Improves Outcomes in Experimental Stroke.

BACKGROUND AND PURPOSE: Human amnion epithelial cells (hAECs) are nonimmunogenic, nontumorigenic, anti-inflammatory cells normally discarded with placental tissue. We reasoned that their profile of biological features, wide availability, and the lack of ethical barriers to their use could make these cells useful as a therapy in ischemic stroke. METHODS: We tested the efficacy of acute (1.5 hours) or delayed (1-3 days) poststroke intravenous injection of hAECs in 4 established animal models of cerebral ischemia. Animals included young (7-14 weeks) and aged mice (20-22 months) of both sexes, as well as adult marmosets of either sex. RESULTS: We found that hAECs administered 1.5 hours after stroke in mice migrated to the ischemic brain via a CXC chemokine receptor type 4-dependent mechanism and reduced brain inflammation, infarct development, and functional deficits. Furthermore, if hAECs administration was delayed until 1 or 3 days poststroke, long-term functional recovery was still augmented in young and aged mice of both sexes. We also showed proof-of-principle evidence in marmosets that acute intravenous injection of hAECs prevented infarct development from day 1 to day 10 after stroke. CONCLUSIONS: Systemic poststroke administration of hAECs elicits marked neuroprotection and facilitates mechanisms of repair and recovery.

Transient visual pathway critical for normal development of primate grasping behavior.

An evolutionary hallmark of anthropoid primates, including humans, is the use of vision to guide precise manual movements. These behaviors are reliant on a specialized visual input to the posterior parietal cortex. Here, we show that normal primate reaching-and-grasping behavior depends critically on a visual pathway through the thalamic pulvinar, which is thought to relay information to the middle temporal (MT) area during early life and then swiftly withdraws. Small MRI-guided lesions to a subdivision of the inferior pulvinar subnucleus (PIm) in the infant marmoset monkey led to permanent deficits in reaching-and-grasping behavior in the adult. This functional loss coincided with the abnormal anatomical development of multiple cortical areas responsible for the guidance of actions. Our study reveals that the transient retino-pulvinar-MT pathway underpins the development of visually guided manual behaviors in primates that are crucial for interacting with complex features in the environment.