A framework and resource for global collaboration in non-human primate neuroscience.

As science and technology evolve, there is an increasing need for promotion of international scientific exchange. Collaborations, while offering substantial opportunities for scientists and benefit to society, also present challenges for those working with animal models, such as non-human primates (NHPs). Diversity in regulation of animal research is sometimes mistaken for the absence of common international welfare standards. Here, the ethical and regulatory protocols for 13 countries that have guidelines in place for biomedical research involving NHPs were assessed with a focus on neuroscience. Review of the variability and similarity in trans-national NHP welfare regulations extended to countries in Asia, Europe and North America. A tabulated resource was established to advance solution-oriented discussions and scientific collaborations across borders. Our aim is to better inform the public and other stakeholders. Through cooperative efforts to identify and analyze information with reference to evidence-based discussion, the proposed key ingredients may help to shape and support a more informed, open framework. This framework and resource can be expanded further for biomedical research in other countries.

Trends and Treatment Approaches for Complications in Neuroscience Experiments with Monkey Species.

Our goal in this manuscript is to advance the assessment and treatment of monkey species in neuroscience research. We hope to begin a discussion and establish baseline data on how complications are identified and treated. We surveyed the neuroscience research community working with monkeys and compiled responses to questions about investigator demographics, assessment of animal wellbeing, treatment choices, and approaches to mitigate risks associated with CNS procedures and promote monkey health and wellbeing. The majority of the respondents had worked with nonhuman primates (NHP) for over 15 y. Identification of procedure-related complications and efficacy of treatment generally rely on common behavioral indices. Treatments for localized inflammatory responses are generally successful, whereas the treatment success for meningitis or meningoencephalitis, abscesses, and hemorrhagic stroke are less successful. Behavioral signs of pain are treated successfully with NSAIDs and opioids. Our future plans are to collate treatment protocols and develop best practices that can be shared across the neuroscience community to improve treatment success rates and animal welfare and therefore science. Human protocols can be used to develop best practices, assess outcomes, and promote further refinements in treatment practices for monkeys to enhance research outcomes.

Miniscope-LFOV: A large-field-of-view, single-cell-resolution, miniature microscope for wired and wire-free imaging of neural dynamics in freely behaving animals.

Imaging large-population, single-cell fluorescent dynamics in freely behaving animals larger than mice remains a key endeavor of neuroscience. We present a large-field-of-view open-source miniature microscope (MiniLFOV) designed for large-scale (3.6 mm × 2.7 mm), cellular resolution neural imaging in freely behaving rats. It has an electrically adjustable working distance of up to 3.5 mm ± 100 µm, incorporates an absolute head orientation sensor, and weighs only 13.9 g. The MiniLFOV is capable of both deep brain and cortical imaging and has been validated in freely behaving rats by simultaneously imaging >1000 GCaMP7s-expressing neurons in the hippocampal CA1 layer and in head-fixed mice by simultaneously imaging ~2000 neurons in the dorsal cortex through a cranial window. The MiniLFOV also supports optional wire-free operation using a novel, wire-free data acquisition expansion board. We expect that this new open-source implementation of the UCLA Miniscope platform will enable researchers to address novel hypotheses concerning brain function in freely behaving animals.

Direct Comparison of Epifluorescence and Immunostaining for Assessing Viral Mediated Gene Expression in the Primate Brain.

Viral vector technologies are commonly used in neuroscience research to understand and manipulate neural circuits, but successful applications of these technologies in non-human primate models have been inconsistent. An essential component to improve these technologies is an impartial and accurate assessment of the effectiveness of different viral constructs in the primate brain. We tested a diverse array of viral vectors delivered to the brain and extraocular muscles of macaques and compared three methods for histological assessment of viral-mediated fluorescent transgene expression: epifluorescence (Epi), immunofluorescence (IF), and immunohistochemistry (IHC). Importantly, IF and IHC identified a greater number of transduced neurons compared to Epi. Furthermore, IF and IHC reliably provided enhanced visualization of transgene in most cellular compartments (i.e., dendritic, axonal, and terminal fields), whereas the degree of labeling provided by Epi was inconsistent and predominantly restricted to somas and apical dendrites. Because Epi signals are unamplified (in contrast to IF and IHC), Epi may provide a more veridical assessment for the amount of accumulated transgene and, thus, the potential to chemogenetically or optogenetically manipulate neuronal activity. The comparatively weak Epi signals suggest that the current generations of viral constructs, regardless of delivered transgene, are not optimized for primates. This reinforces an emerging viewpoint that viral vectors tailored for the primate brain are necessary for basic research and human gene therapy.

Unified ethical principles and an animal research 'Helsinki' declaration as foundations for international collaboration.

Ethical frameworks are the foundation for any research with humans or nonhuman animals. Human research is guided by overarching international ethical principles, such as those defined in the Helsinki Declaration by the World Medical Association. However, for nonhuman animal research, because there are several sets of ethical principles and national frameworks, it is commonly thought that there is substantial variability in animal research approaches internationally and a lack of an animal research 'Helsinki Declaration', or the basis for one. We first overview several prominent sets of ethical principles, including the 3Rs, 3Ss, 3Vs, 4Fs and 6Ps. Then using the 3Rs principles, originally proposed by Russell & Burch, we critically assess them, asking if they can be Replaced, Reduced or Refined. We find that the 3Rs principles have survived several replacement challenges, and the different sets of principles (3Ss, 3Vs, 4Fs and 6Ps) are complementary, a natural refinement of the 3Rs and are ripe for integration into a unified set of principles, as proposed here. We also overview international frameworks and documents, many of which incorporate the 3Rs, including the Basel Declaration on animal research. Finally, we propose that the available animal research guidance documents across countries can be consolidated, to provide a similar structure as seen in the Helsinki Declaration, potentially as part of an amended Basel Declaration on animal research. In summary, we observe substantially greater agreement on and the possibility for unification of the sets of ethical principles and documents that can guide animal research internationally.

Next-generation brain observatories.

We propose centralized brain observatories for large-scale recordings of neural activity in mice and non-human primates coupled with cloud-based data analysis and sharing. Such observatories will advance reproducible systems neuroscience and democratize access to the most advanced tools and data.

Optogenetic activation of the inhibitory nigro-collicular circuit evokes contralateral orienting movements in mice.

We report that increasing inhibition from the basal ganglia (BG) to the superior colliculus (SC) through the substantia nigra pars reticulata (nigra) using in vivo optogenetic activation of GABAergic terminals in mice produces contralateral orienting movements. These movements are unexpected because decreases, and not increases, in nigral activity are generally associated with the initiation of orienting movements. We found that, in slice recordings, the same optogenetic stimulation of nigral terminals producing movements in vivo evokes post-inhibitory rebound depolarization followed by Na+ spikes in SC output neurons. Moreover, blocking T-type Ca2+ channels in slices prevent post-inhibitory rebound and subsequent Na+ spiking in SC output neurons and also reduce the likelihood of contralateral orienting in vivo. On the basis of these results, we propose that, in addition to the permissive role, the BG may play an active role in the generation of orienting movements in mice by driving post-inhibitory rebound depolarization in SC output neurons.

Causal role for the primate superior colliculus in the computation of evidence for perceptual decisions.

Trained monkeys performed a two-choice perceptual decision-making task in which they reported the perceived orientation of a dynamic Glass pattern, before and after unilateral, reversible, inactivation of a brainstem area-the superior colliculus (SC)-involved in preparing eye movements. We found that unilateral SC inactivation produced significant decision biases and changes in reaction times consistent with a causal role for the primate SC in perceptual decision-making. Fitting signal detection theory and sequential sampling models to the data showed that SC inactivation produced a decrease in the relative evidence for contralateral decisions, as if adding a constant offset to a time-varying evidence signal for the ipsilateral choice. The results provide causal evidence for an embodied cognition model of perceptual decision-making and provide compelling evidence that the SC of primates (a brainstem structure) plays a causal role in how evidence is computed for decisions-a process usually attributed to the forebrain.

Tuned inhibition in perceptual decision-making circuits can explain seemingly suboptimal confidence behavior.

Current dominant views hold that perceptual confidence reflects the probability that a decision is correct. Although these views have enjoyed some empirical support, recent behavioral results indicate that confidence and the probability of being correct can be dissociated. An alternative hypothesis suggests that confidence instead reflects the magnitude of evidence in favor of a decision while being relatively insensitive to the evidence opposing the decision. We considered how this alternative hypothesis might be biologically instantiated by developing a simple neural network model incorporating a known property of sensory neurons: tuned inhibition. The key idea of the model is that the level of inhibition that each accumulator unit receives from units with the opposite tuning preference, i.e. its inhibition 'tuning', dictates its contribution to perceptual decisions versus confidence judgments, such that units with higher tuned inhibition (computing relative evidence for different perceptual interpretations) determine perceptual discrimination decisions, and units with lower tuned inhibition (computing absolute evidence) determine confidence. We demonstrate that this biologically plausible model can account for several counterintuitive findings reported in the literature where confidence and decision accuracy dissociate. By comparing model fits, we further demonstrate that a full complement of behavioral data across several previously published experimental results-including accuracy, reaction time, mean confidence, and metacognitive sensitivity-is best accounted for when confidence is computed from units without, rather than units with, tuned inhibition. Finally, we discuss predictions of our results and model for future neurobiological studies. These findings suggest that the brain has developed and implements this alternative, heuristic theory of perceptual confidence computation by relying on the diversity of neural resources available.

Variable Statistical Structure of Neuronal Spike Trains in Monkey Superior Colliculus.

Popular models of decision-making propose that noisy sensory evidence accumulates until reaching a bound. Behavioral evidence as well as trial-averaged ramping of neuronal activity in sensorimotor regions of the brain support this idea. However, averaging activity across trials can mask other processes, such as rapid shifts in decision commitment, calling into question the hypothesis that evidence accumulation is encoded by delay period activity of individual neurons. We mined two sets of data from experiments in four monkeys in which we recorded from superior colliculus neurons during two different decision-making tasks and a delayed saccade task. We applied second-order statistical measures and spike train simulations to determine whether spiking statistics were similar or different in the different tasks and monkeys, despite similar trial-averaged activity across tasks and monkeys. During a motion direction discrimination task, single-trial delay period activity behaved statistically consistent with accumulation. During an orientation detection task, the activity behaved superficially like accumulation, but statistically consistent with stepping. Simulations confirmed both findings. Importantly, during a simple saccade task, with similar trial-averaged activity, neither process explained spiking activity, ruling out interpretations based on differences in attention, reward, or motor planning. These results highlight the need for exploring single-trial spiking dynamics to understand cognitive processing and raise the interesting hypothesis that the superior colliculus participates in different aspects of decision-making depending on task differences.SIGNIFICANCE STATEMENT How are decisions based on sensory information transformed into actions? We report that single-trial neuronal activity dynamics in the superior colliculus of monkeys show differences in decision-making tasks depending on task idiosyncrasies and requirements and despite similar trial-averaged ramping activity. These results highlight the importance of exploring single-trial spiking dynamics to understand cognitive processing and raise the interesting hypothesis that the superior colliculus participates in different aspects of decision-making depending on task requirements.

Unraveling circuits of visual perception and cognition through the superior colliculus.

The superior colliculus is a conserved sensorimotor structure that integrates visual and other sensory information to drive reflexive behaviors. Although the evidence for this is strong and compelling, a number of experiments reveal a role for the superior colliculus in behaviors usually associated with the cerebral cortex, such as attention and decision-making. Indeed, in addition to collicular outputs targeting brainstem regions controlling movements, the superior colliculus also has ascending projections linking it to forebrain structures including the basal ganglia and amygdala, highlighting the fact that the superior colliculus, with its vast inputs and outputs, can influence processing throughout the neuraxis. Today, modern molecular and genetic methods combined with sophisticated behavioral assessments have the potential to make significant breakthroughs in our understanding of the evolution and conservation of neuronal cell types and circuits in the superior colliculus that give rise to simple and complex behaviors.

International primate neuroscience research regulation, public engagement and transparency opportunities.

Scientific excellence is a necessity for progress in biomedical research. As research becomes ever more international, establishing international collaborations will be key to advancing our scientific knowledge. Understanding the similarities in standards applied by different nations to animal research, and where the differences might lie, is crucial. Cultural differences and societal values will also contribute to these similarities and differences between countries and continents. Our overview is not comprehensive for all species, but rather focuses on non-human primate (NHP) research, involving New World marmosets and Old World macaques, conducted in countries where NHPs are involved in neuroimaging research. Here, an overview of the ethics and regulations is provided to help assess welfare standards amongst primate research institutions. A comparative examination of these standards was conducted to provide a basis for establishing a common set of standards for animal welfare. These criteria may serve to develop international guidelines, which can be managed by an International Animal Welfare and Use Committee (IAWUC). Internationally, scientists have a moral responsibility to ensure excellent care and welfare of their animals, which in turn, influences the quality of their research. When working with animal models, maintaining a high quality of care ("culture of care") and welfare is essential. The transparent promotion of this level of care and welfare, along with the results of the research and its impact, may reduce public concerns associated with animal experiments in neuroscience research.

An Open Resource for Non-human Primate Optogenetics.

Optogenetics has revolutionized neuroscience in small laboratory animals, but its effect on animal models more closely related to humans, such as non-human primates (NHPs), has been mixed. To make evidence-based decisions in primate optogenetics, the scientific community would benefit from a centralized database listing all attempts, successful and unsuccessful, of using optogenetics in the primate brain. We contacted members of the community to ask for their contributions to an open science initiative. As of this writing, 45 laboratories around the world contributed more than 1,000 injection experiments, including precise details regarding their methods and outcomes. Of those entries, more than half had not been published. The resource is free for everyone to consult and contribute to on the Open Science Framework website. Here we review some of the insights from this initial release of the database and discuss methodological considerations to improve the success of optogenetic experiments in NHPs.

Adeno-Associated Virus Capsid-Promoter Interactions in the Brain Translate from Rat to the Nonhuman Primate.

Recently, we established an adeno-associated virus (AAV9) capsid-promoter interaction that directly determined cell-specific gene expression across two synthetic promoters, Cbh and CBA, in the rat striatum. These studies not only expand this capsid-promoter interaction to include another promoter in the rat striatum but also establish AAV capsid-promoter interactions in the nonhuman primate brain. When AAV serotype 9 (AAV9) vectors were injected into the rat striatum, the minimal synthetic promoter JetI drove green fluorescent protein (GFP) gene expression predominantly in oligodendrocytes. However, similar to our previous findings, the insertion of six alanines into VP1/VP2 of the AAV9 capsid (AAV9AU) significantly shifted JetI-driven GFP gene expression to neurons. In addition, previous retrograde tracing studies in the nonhuman primate brain also revealed the existence of a capsid-promoter interaction. When rAAV2-Retro vectors were infused into the frontal eye field (FEF) of rhesus macaques, local gene expression was prominent using either the hybrid chicken beta actin (CAG) or human synapsin (hSyn) promoters. However, only the CAG promoter, not the hSyn promoter, led to gene expression in the ipsilateral claustrum and contralateral FEF. Conversely, infusion of rAAV2-retro-hSyn vectors, but not rAAV2-retro-CAG, into the macaque superior colliculus led to differential and selective retrograde gene expression in cerebellotectal afferent cells. Clearly, this differential promoter/capsid expression profile could not be attributed to promoter inactivation from retrograde transport of the rAAV2-Retro vector. In summary, we document the potential for AAV capsid/promoter interactions to impact cell-specific gene expression across species, experimental manipulations, and engineered capsids, independent of capsid permissivity.

Using rAAV2-retro in rhesus macaques: Promise and caveats for circuit manipulation.

BACKGROUND: Recent genetic technologies such as opto- and chemogenetics allow for the manipulation of brain circuits with unprecedented precision. Most studies employing these techniques have been undertaken in rodents, but a more human-homologous model for studying the brain is the nonhuman primate (NHP). Optimizing viral delivery of transgenes encoding actuator proteins could revolutionize the way we study neuronal circuits in NHPs. NEW METHOD: rAAV2-retro, a popular new capsid variant, produces robust retrograde labeling in rodents. Whether rAAV2-retro's highly efficient retrograde transport would translate to NHPs was unknown. Here, we characterized the anatomical distribution of labeling following injections of rAAV2-retro encoding opsins or DREADDs in the cortico-basal ganglia and oculomotor circuits of rhesus macaques. RESULTS: rAAV2-retro injections in striatum, frontal eye field, and superior colliculus produced local labeling at injection sites and robust retrograde labeling in many afferent regions. In every case, however, a few brain regions with well-established projections to the injected structure lacked retrogradely labeled cells. We also observed robust terminal field labeling in downstream structures. COMPARISON WITH EXISTING METHOD(S): Patterns of labeling were similar to those obtained with traditional tract-tracers, except for some afferent labeling that was noticeably absent. CONCLUSIONS: rAAV2-retro promises to be useful for circuit manipulation via retrograde transduction in NHPs, but caveats were revealed by our findings. Some afferently connected regions lacked retrogradely labeled cells, showed robust axon terminal labeling, or both. This highlights the importance of anatomically characterizing rAAV2-retro's expression in target circuits in NHPs before moving to manipulation studies.

Superior colliculus signals decisions rather than confidence: analysis of single neurons.

Recent findings indicate that monkeys can report their confidence in perceptual decisions and that this information is encoded in neurons involved in making decisions, including the lateral intraparietal area (LIP) and the supplementary eye field (SEF). A key issue to consider when studying confidence is that decision accuracy often correlates with confidence reports; when we are performing well, we generally feel more confident. Expanding on work performed in humans, we designed a novel task for monkeys that dissociates perceptual information leading to decisions from perceptual information leading to confidence reports. Using this task, we recently showed that decoded ensemble activity recorded from the superior colliculus (SC) reflected decisions rather than confidence reports. However, our previous population level analysis collapsed over multiple SC neuronal types and therefore left open the possibility that first, individual discharge rates might encode information related to decision confidence, and second, different neuronal cell types within the SC might signal decision confidence independently of decision accuracy. We found that when decision accuracy and decision confidence covaried, modulation occurred primarily in neurons with prelude activity (buildup neurons). However, isolating decision confidence from decision accuracy uncovered that only a few, primarily buildup neurons showed signals correlating uniquely with decision confidence and the effect sizes were very small. Based on this work and our previous work using decoding methods, we conclude that neuronal signals for decision confidence, independent of decision accuracy, are unlikely to exist at the level of single or populations of neurons in the SC. Our results together with other recent work call into question normative models of confidence based on the optimal readout of decision signals. NEW & NOTEWORTHY Models of decision confidence suggest that our sense of confidence is an optimal readout of perceptual decision signals. Here, we report that a subcortical area, the superior colliculus (SC), contains neurons with activity that signal decisions and confidence in a task in which decision accuracy and confidence covary, similar to area lateral intraparietal area in cortex. The signals from SC occur primarily in the neurons with prelude activity (buildup neurons). However, in a task that dissociates decision accuracy from decision confidence, we find that only a few individual neurons express unique signals of confidence. These results call into question normative models of confidence based on optimal readout of perceptual decision signals.

Parvalbumin and GABA Microcircuits in the Mouse Superior Colliculus.

The mammalian superior colliculus (SC) is a sensorimotor midbrain structure responsible for orienting behaviors. Although many SC features are known, details of its intrinsic microcircuits are lacking. We used transgenic mice expressing reporter genes in parvalbumin-positive (PV+) and gamma aminobutyric acid-positive (GABA+) neurons to test the hypothesis that PV+ neurons co-localize GABA and form inhibitory circuits within the SC. We found more PV+ neurons in the superficial compared to the intermediate SC, although a larger percentage of PV+ neurons co-expressed GABA in the latter. Unlike PV+ neurons, PV+/GABA+ neurons showed predominantly rapidly inactivating spiking patterns. Optogenetic activation of PV+ neurons revealed direct and feedforward GABAergic inhibitory synaptic responses, as well as excitatory glutamatergic synapses. We propose that PV+ neurons in the SC may be specialized for a variety of circuit functions within the SC rather than forming a homogeneous, GABAergic neuronal subtype as they appear to in other regions of the brain.

Paradoxical Decision-Making: A Framework for Understanding Cognition in Parkinson's Disease.

People with Parkinson's disease (PD) show impaired decision-making when sensory and memory information must be combined. This recently identified impairment results from an inability to accumulate the proper amount of information needed to make a decision and appears to be independent of dopamine tone and reinforcement learning mechanisms. Although considerable work focuses on PD and decisions involving risk and reward, in this Opinion article we propose that the emerging findings in perceptual decision-making highlight the multisystem nature of PD, and that unraveling the neuronal circuits underlying perceptual decision-making impairment may help in understanding other cognitive impairments in people with PD. We also discuss how a decision-making framework may be extended to gain insights into mechanisms of motor impairments in PD.

Superior colliculus neuronal ensemble activity signals optimal rather than subjective confidence.

Recent studies suggest that neurons in sensorimotor circuits involved in perceptual decision-making also play a role in decision confidence. In these studies, confidence is often considered to be an optimal readout of the probability that a decision is correct. However, the information leading to decision accuracy and the report of confidence often covaried, leaving open the possibility that there are actually two dissociable signal types in the brain: signals that correlate with decision accuracy (optimal confidence) and signals that correlate with subjects' behavioral reports of confidence (subjective confidence). We recorded neuronal activity from a sensorimotor decision area, the superior colliculus (SC) of monkeys, while they performed two different tasks. In our first task, decision accuracy and confidence covaried, as in previous studies. In our second task, we implemented a motion discrimination task with stimuli that were matched for decision accuracy but produced different levels of confidence, as reflected by behavioral reports. We used a multivariate decoder to predict monkeys' choices from neuronal population activity. As in previous studies on perceptual decision-making mechanisms, we found that neuronal decoding performance increased as decision accuracy increased. However, when decision accuracy was matched, performance of the decoder was similar between high and low subjective confidence conditions. These results show that the SC likely signals optimal decision confidence similar to previously reported cortical mechanisms, but is unlikely to play a critical role in subjective confidence. The results also motivate future investigations to determine where in the brain signals related to subjective confidence reside.