John H. Reynolds - US grants

The long-range goal of this research is to understand the neural mechanisms of selective visual attention at the level of the individual neuron and the cortical circuit, and to determine how these mechanisms affect perception and behavior. The conceptual starting point is the observation that the brain is limited in the amount of visual information it can process at any moment in time. For instance, when observers are asked to identify the objects in a briefly presented scene, they become less accurate as the number of objects increases. The proposed research will use a combination of visual psychophysics and single-unit physiology to test two alternative explanations for this capacity limitation, and to investigate the neural mechanisms that enable the brain to select behaviorally relevant stimuli from among irrelevant distracters. Insights from the proposed research are expected to help in understanding, diagnosis and treatment of neuropsychological disorders in which attentional mechanisms fail, such as neglect, Balint's syndrome, ADHD, and attentional aspects of autism.

[unreadable] DESCRIPTION (provided by applicant): The proposed projects involve making single-unit recording studies in the awake monkey to test a simple cortical circuit model of attention. If supported empirically, the model promises to provide a major simplification in our understanding of the neural mechanisms of spatial attention. At the end of the proposed course of research, we expect to have achieved important and profoundly simplifying insights into the neural mechanisms of attention. We will have characterized key contrast-dependent response properties of V4, which itself will constitute an important step forward (Aim 1). We will have tested whether these contrast-dependent response properties in Area V4 mirror those of V1 (Aim 1). Understanding any differences that may exist between contrast modulation in V4 and V 1 is important. To the extent that they are similar, this will allow us to establish a mechanistic link between these two important visual processing areas, and to broaden the significance of models of processing in V 1. We will have tested whether spatial attention modulates center-surround interactions (Aim 2), which has major ramifications for our understanding of earlier studies. For example, single-unit recording studies in V2, V4, MT and TEO have established that when two stimuli appear together within a neuron's classical RF, the response is driven preferentially by the attended stimulus. The proposed studies will determine whether this pattern occurs as a result of biasing suppressive center-surround interactions at earlier stages. If so, this will support the simple model advanced in this proposal. If not, this would strongly suggest that the selection occurs at the first stage where RFs are large enough to encompass the two stimuli. Finally, we will have measured the relationship between contrast-dependent center-surround modulations and attentional selection (Aim 3), providing the strongest test yet of the proposal that attention operates by increasing the effective contrast of the attended stimulus. Taken together, the proposed research promises to provide a much deeper understanding of the cortical circuits that mediate attentional selection, than has been achieved to date. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): A central observation that has driven modern thinking about vision is that the visual system decomposes stimuli into their constituent features, represented by neurons with different feature selectivities. How the signals carried by these feature selective neurons are integrated into coherent object representations is unknown and stands as one of the central unsolved questions in neurobiology. Insight into the mechanisms that solve this problem can be gained by carefully analyzing the temporal properties of perceptual integration at the psychophysical and neuronal levels. We will pursue this strategy, focusing on a simple and well-defined case of feature integration: the conjoining of two of the most basic and well studied features in vision: color and orientation. The central objective of the current proposal is to understand how the visual system conjoins these features. This objective will be met in the following specific aims. Aim 1. Compare the temporal resolution of the visual system for color, orientation and conjunctions of these two features, at the perceptual level and in BOLD in humans, and at the neuronal level in monkeys. Aim 2. Characterize the temporal dynamics of tuning for color and orientation, their dependence on luminance contrast, and quantify any changes in these properties under naturalistic visual stimulation. Aim 3. Test whether spatial attention plays a role in the integration of color and orientation. Relevance: Our current lack of knowledge about the mechanisms of feature integration represents a significant gap in our understanding of the pathology of disorders in which the perception of objects is impaired, such as simultagnosia, prosopagnosia, integrative visual agnosia, Balint's syndrome, and object- centered impairments associated with neglect. Filling this gap will provide understanding that will help in the interpretation and treatment of these and related disorders. The proposed experiments will close this gap by (1) characterizing the temporal resolution of the neural mechanisms that mediate feature integration, (2) developing and testing mathematically rigorous models of the neural computations that mediate a simple and well defined form of feature integration, and (3) determining whether or not attention plays a role in the formation of feature conjunctions.

DESCRIPTION (provided by applicant): In the proposed research, we will use a newly developed primate optogenetic tool, which enables us to create reversible micro lesions within the neocortex of awake, behaving monkeys. Using this tool, we will test and quantify the role of macaque Area V4, an intermediate stage of processing within the ventral visual processing stream. In Aim 1, we will examine how this new tool modulates the gain of neuronal responses, and will quantify the region of activation as a function of illumination level. Area V4 has a patchy organization, with some regions being selective for color, and others selective for orientation. In Aim 2, we will test the role of V4 in the perception of color and of orientation, by having monkeys make fine discriminations of color and orientation, while we inactivate patches color- or orientation-selective neurons. We will examine whether activating the neurons tuned for color impairs color judgments, and not orientation judgments, and whether activating the neurons tuned for orientation impairs orientation judgments, and not color judgments. We will also examine whether this impairment is greater when we inactivate neurons tuned for the discriminanda or, alternatively, tuned such that the steep part of the tuning curve is suited to discriminate among discriminanda. In Aim 3, we will use optogenetic micro lesions to test whether V4 plays a causal role in mediating attentional selection. The data we collect will provide the most direct evidence for or against the proposition that V4 plays a causal role in the perception of color and of orientation, and whether it plays a causal role in attentional selection. In addition, the proposed research will demonstrate the use of this new optogenetic tool, and we will make it publicly available, so that other researchers working in primates and in other species, can use this tool to test theories of perception, cognition and action.

DESCRIPTION (provided by applicant): Our understanding of the neural mechanisms underlying action, perception and cognition has been limited by the lack of tools to modulate the activity of specific neural circuits in the awake, behaving animal. This technical hurdle has, in turn, limited our understanding of devastating disorders of the nervous system that result from dysfunction of neural circuits. The proposed research addresses this challenge by developing a new set of optogenetic techniques that will make it possible to selectively manipulate the activity of projection neurons with precise temporal control in the brain of the behaving non-human primate. We plan to inject viral vectors, specifically designed to be taken up by axon terminals, into the brain in order to get projection neurons to express the light-sensitive channels channelrhodopsin-2 and archaerhodopsin-3. We will then be able to selectively activate or inactivate projection neurons using light introduced into the brain with specially designed optitrodes. Our proposal focuses on projections from primary visual cortex (V1) to the superficial layers of the superior colliculus and V1 neurons projecting to the secondary visual area (V2), taking advantage of the well- defined retinotopic maps in these regions, and using physiological, behavioral, and histological methods to measure our ability to selectively manipulate the activity of projection neurons. The results of these studies are likely to have a large impact, because the tools and techniques developed in this research program will have wide applicability for those studying the relationships between functional neural networks in the CNS and primate behavior. They will also provide important tests of the feasibility of optical and virus-based methods as possible therapeutic approaches in CNS disorders. PUBLIC HEALTH RELEVANCE: Understanding the functional role of particular neural connections is crucial for unraveling the etiology of neuropsychological disorders such as neglect, Balint's syndrome, visual agnosia, schizophrenia, ADHD and autism. The goal of this project is to establish new techniques for selectively manipulating the activity of projection neurons in the behaving non-human primate under precise temporal control using combinations of physiological, molecular, and genetic methods. The results from our studies will demonstrate powerful new tools for studying the functional relationships between neural networks and primate behavior, and provide important tests of the feasibility of optical and virus-based methods as possible therapeutic approaches in CNS disorders.

PROJECT SUMMARY The long-term goal of this project is to model human neurodegenerative diseases in marmosets via gene- editing in embryonic stem cells (ESCs). The mouse system is a powerful tool for medical research due to the ability to manipulate the mouse genome. However, considerable anatomical, physiological, cognitive, and behavioral differences between mice and humans limit the degree to which insights from mouse models shed light on human diseases. This is reflected in the high number of failed clinical trails for drugs that were effective in treating mouse models of human disease. Several lines of evidence suggest that the marmoset represents an improved animal system for studying a range of human diseases, including stroke and age-associated neurodegenerative diseases such as Alzheimer's disease (AD). Marmosets are the shortest-lived of the anthropoid primates (average lifespan of 5?7 years compared with 25 years for the rhesus macaque) and exhibit age-related changes that are similar to those seen in humans, including ?-amyloid deposition in the cerebral cortex, loss of cholinergic innervation, and reduced neurogenesis, as observed in AD. In addition, marmosets are highly social and communicative and have demonstrated the capacity to learn sophisticated cognitive behaviors. Therefore, marmosets represent an ideal genetic platform for generating models of neurodegenerative diseases that more accurately reflect the human condition and enable the testing of potential autologous (the-same-species) stem cell-based regenerative therapies. Initial efforts will focus on generating a marmoset model of AD. The recent emergence of gene-editing and stem-cell technologies in primates pave the way toward generating marmoset disease models, but improvements in both areas are necessary to make this approach viable. Here, both conventional homologous recombination and CRISPR/Cas9 genome-editing technologies will be employed to modify marmoset ESCs. As genetic evidence demonstrates that mutations in the amyloid precursor protein (APP) gene result in increased ?-amyloid production, the formation of plaques, and cognitive impairment, the marmoset APP will be edited to carry human point mutations. Genetic tools for studying neuronal cell type-specific circuits underlying cognitive impairment and neuropathology in AD will also be generated by inserting a Cre recombinase cassette into 3' end non-translated regions of the parvalbumin and choline acetyltransferase genes. These Cre driver lines will enable the visualization and functional manipulation of these cell types. Successful completion of the proposed Aims will generate a greatly improved animal model of AD, enable testing of stem cell-based regenerative methods for treating AD, and pave the way toward applying these genetic tools for analyzing neuronal circuitry of healthy brains. Establishing gene-editing in marmoset ESCs will also enable the development of additional primate models of human diseases, providing critical experimental resources for research supported by multiple NIH Institutes (e.g., NINDS, NIA, NIMH, NEI, NIAAA, NIDA, NICHG, NIGMS).

Project Summary/Abstract The ability for an animal or human to perceive a subtle environmental stimulus is not a fixed parameter. Rather, perceptual thresholds fluctuate with changes in arousal, attention, and expectation. Similarly, individual neurons within the visual cortex exhibit variable responses when repeatedly presented the same stimulus, an observation widely thought to contribute to perceptual variability. However, injection of identical noisy currents evokes highly precise spike trains, indicating that these fluctuations are not due to a noisy spiking mechanism. Instead, it largely reflects moment-by-moment synaptic input from the cortical network. These network fluctuations are reflected in local field potentials (LFPs). Here, new computational methods have been used to show for the first time that spontaneous fluctuations are organized into traveling waves in awake, behaving primates. These methods enable tracking of traveling waves on a moment-by-moment basis, without trial averaging analyses. Spontaneous waves create periods of both elevated and suppressed spiking activity, and preliminary data indicate that they modulate stimulus-evoked spiking responses and perceptual sensitivity in a visual detection task. Thus, the assembled team is well positioned to understand the role of neocortical traveling waves in perception and propose three Aims. Aim 1: Test whether spontaneous activity in awake marmoset MT and V1 is organized into traveling waves. Utah arrays will be implanted in marmoset area MT and V1 to record spikes and LFPs while the monkey fixates a blank screen. Network fluctuations will be detected to test the hypothesis that spontaneous spiking activity generates traveling waves that can be detected in the LFP, and the phase of LFP fluctuations reflect periods of depolarization and hyperpolarization. Aim 2: Develop a spiking network model linking LFP waves, spiking activity and perception. A preliminary computational model has been developed that accounts for spike-LFP relationships observed in experimental data. Here, the model will be extended to quantitatively match properties of observed traveling waves, and then used to generate testable predictions about how the phase of LFP waves affects spiking probability, stimulus-evoked responses, and perceptual sensitivity (the latter by extending the model within an ideal observer framework). Aim 3: Determine the impact of traveling waves on sensory perception. The model predicts that spontaneous traveling waves will both increase and decrease the gain of a stimulus-evoked response, depending on wave phase. To test this, spontaneous waves will be recorded within MT and V1 as marmosets attempt to detect a faint visual stimulus. This will also allow researchers to test the model prediction that wave phase regulates perceptual sensitivity. Together, these analyses will help characterize the contributions of spontaneous traveling waves to cortical variability and perception, information critical for understanding brain disorders associated with failures in perception and attention, such as autism, schizophrenia, and Alzheimer?s disease.

Project Summary  Despite the enormous complexity of the brain, it is becoming increasingly apparent that structures like the  cerebral cortex are modular, relying on a set of canonical computations that occur across brain regions and  modalities to mediate perception, cognition and behavior.  One important example of a canonical computation  is the summation of various driving, contextual, and modulatory neuronal inputs to yield spiking output. The  question of how cortical networks integrate these inputs and transform them into spiking outputs of individual  neurons is of central importance to neuroscience.  A significant challenge to understanding these computations  is that each neuron is embedded within a larger circuit of neurons, each modulating one another?s activity.  So,  understanding how a particular neuron responds to input necessarily involves understanding the larger circuit.   Recent optogenetic studies have found different patterns of input summation in mouse vs. monkey V1.  Recently developed theoretical models have produced specific predictions about the differences in network  circuitry that can lead to differences in summation, and predict how summation non-­linearities depend on  inputs to the network. The proposed research will test these predictions and seek to understand these circuit  computations using a combination of theoretical work and optogenetic modulation of circuits in mouse and  monkey.  Aim 1: Varying E and I optogenetic stimulation and visual contrast independently to measure  spike response summation to multiple inputs. In this Aim, theoretical models of input summation across  varying cortical circuit regimes will be developed, and recently developed optogenetic tools will be used in  awake mouse and monkey V1 to test predictions generated by these models and identify the corresponding  regimes. The optogenetic tools include a new viral strategy that directs expression of different opsins to  inhibitory vs. excitatory neocortical neurons in the macaque. Simultaneous and independent activation of E and  I and the visual stimulus, all within this theoretical framework, will enable us to test whether observed  differences in summation properties reflect fundamental species differences or reflect a common computation  operating in different parameter regimes.  Aim 2: Determine the circuit elements controlling dynamics of  cortical network responses using dynamic optogenetic stimulation. In this Aim, experiments using  dynamic optogenetic and visual stimulation patterns and theoretical analysis of the models with dynamic inputs  will be used to elucidate the temporal dynamics of summation. Aim 3: Determine if different inhibitory  subclasses control different aspects of input integration. Different inhibitory subclasses will be stimulated  optogenetically to decipher their respective roles in input summation. Taken together, these Aims will help  define the roles played by excitatory and inhibitory neurons in mediating summation of neuronal inputs to yield  spiking output. This information will be critical for understanding brain disorders associated with failures in  perception and attention, as is seen with autism, schizophrenia, and Alzheimer?s disease.

PROJECT SUMMARY/ABSTRACT An unfortunate consequence of increased human lifespan is that aging-related neurodegenerative diseases such as Alzheimer?s disease (AD) are a growing burden. The single greatest risk factor for cognitive decline and AD is aging, yet we lack an understanding of how aging predisposes the brain to these consequences. Synaptic alterations that correlate with cognitive dysfunction are prevalent in both normal and pathological aging, though they are distinct from one another. In normal aging, subtle synaptic changes that affect the number of functional glutamatergic receptors lead to a decrease in synaptic efficacy and result in cognitive decline. In AD, synaptic loss precedes cell death and is the neuropathological feature of AD that correlates most strongly with cognitive dysfunction. Synaptic changes are unmistakably an important feature of normal aging and pathological neurodegeneration, and the clear links to cognitive decline underscore the importance of simultaneously investigating these features of aging. Non-human primates (NHPs) have similar neuroanatomy, neurophysiology, and cognitive abilities to humans, and also can develop the hallmark neuropathologies of AD; Tau tangles and ?-amyloid plaques. The proposed research develops the common marmoset (Callithrix jacchus) as a NHP model for simultaneously investigating aging-related cognitive decline and synaptic alterations. Compared to macaque monkeys with long lifespans (25-40 years), marmosets have a relatively short average lifespan of 9-10 years, enabling their use in longitudinal studies of aging. In Aim 1, aging-related deficits in marmosets? performance of a hippocampus-dependent memory task, the Delayed Recognition Span Task (DRST), will be determined. Performance of this task declines with normal aging in humans and macaques and patients with mild AD are further impaired on the DRST compared to healthy elderly controls. This task has been developed for marmosets using an automated touch screen system within the animal?s home cage. This Aim will test the hypothesis that aged animals exhibit impaired memory capacity on the task, reflecting aging-related cognitive decline. In Aim 2, non-invasive positron emission tomography (PET) imaging will be used to measure synaptic alterations in the hippocampus of the marmosets used for cognitive testing in Aim 1. To do this, a PET tracer that selectively binds to AMPA receptors will be used. This Aim will test the hypothesis that aged marmosets have decreased tracer uptake in the hippocampus, reflecting decreased AMPA receptor density, compared to younger animals. Further, there will be a strong, positive correlation between AMPA receptor density and cognitive performance, measured via the DRST in Aim 1. Establishing this experimental platform for the simultaneous tracking of aging-related changes in both hippocampal-dependent memory and AMPA receptor density will enable future longitudinal investigations to elucidate the time course of synaptic changes and cognitive decline in the brains of marmosets. This could lead to novel ways to predict onset of neurodegeneration before clinical symptoms arise.