Martha E. Bickford - US grants

[unreadable] DESCRIPTION (provided by applicant): The long term goal of the proposed experiments is to determine the causes of attention impairment during disease. However, it is first necessary to understand the organization of circuits that may contribute to attention mechanisms in the normal brain. Many studies have implicated the pulvinar nucleus as an area that may regulate visual attention. We propose to study the rodent lateral posterior (LP) nucleus, a region comparable to inferior regions of the primate pulvinar nucleus. We will use a combination of tract tracing, immunocytochemistry, electron microscopy and in vitro electrophysiology to examine the synaptic organization and functional properties of LP neurons. We propose that the rat LP nucleus contains at least 2 subdivisions that are characterized by distinct synaptic inputs, axonal projection patterns, and cellular firing properties. Regular spiking cells in the caudal LP nucleus are predicted to receive tectal input and project to the striatum. The synaptic arrangements of tectothalamic terminals, coupled with the linear firing properties of caudal LP neurons, may serve to accurately transfer signals related to the movement of visual stimuli. This circuitry may underlie the coordination of visually guided movements. Clustered spiking cells in the rostral LP nucleus are predicted to receive input from layer 5 of the visual cortex and in turn project to cortical layer 1. The synaptic arrangements of layer 5 corticothalamic terminals, coupled with the non-linear firing properties of rostral LP neurons, may serve to amplify and synchronize cortical activity, providing a substrate for the selective enhancement of visual signals. A thorough understanding of the firing characteristics of LP neurons and the organization and dynamic properties of their synaptic connections is necessary to begin to formulate reasonable hypotheses regarding the function of the rat LP nucleus, and ultimately the human pulvinar nucleus. [unreadable] [unreadable]

LAY ABSTRACT Principal Investigator: Bickford, Martha Proposal Number: IBN- 9728089 PARALLEL CORTICAL PATHWAYS THROUGH THE PULVINAR COMPLEX Most sensory signals must pass through the dorsal thalamus before they are consciously recognized in the cortex. It has been known for a number of years that sensory signals from peripheral receptors are segregated, and travel in parallel pathways through the relay nuclei of the dorsal thalamus, to primary sensory areas of cortex. The study of parallel pathways in sensory system, particularly in the visual system, has provided a key to understanding how the brain codes the wide variety of incoming information. The realization that features such as color, motion, or contrast are coded by separate subcortical cell types has provided a valuable conceptual framework for studies of how visual signals are perceived by the brain. This proposal tests the hypothesis that similar parallel pathways exist in areas of the thalamus that associate information from a variety of cortical areas. In this case, the proposed parallel pathways originate in cortex, and remain segregated in the thalamus through synaptic contacts on distinct classes of thalamocortical neurons, which project to different cortical laminae. This idea will be tested in the pulvinar and lateral posterior nuclei, which receive input from two types of layer V cortical cells, and contain at least two morphologically distinct types of thalamocortical cells. It is proposed that separate cortical axons contact separate classes of thalamocortical cells that project to different cortical lamina. The proposed anatomical experiments, which involve the injection of neuroanatomical tracers and irnmunocytochemistry, wi ll test the concept that parallel pathways are utilized in the processing of internal cortical signals. It is anticipated that, similar to the study of parallel sensory pathways, this information will provide a fruitful starting point for deciphering the function of the pulvinar and lateral posterior nuclei.

[unreadable] DESCRIPTION (provided by the applicant): Parallel visual pathways from the retina to the cortex, via the lateral geniculate nucleus (LGN) or via the superior colliculus (SC) and pulvinar nucleus, likely serve distinct functions in the coding of form, movement, and spatial location signals. In the LGN, further segregations of anatomically and physiologically distinct visual pathways have been identified and extensively characterized. Likewise, studies in a variety of species have noted the existence of multiple pathways from the SC to the thalamus, although these pathways are largely uncharacterized, and their functions remain unknown. The tree shrew, with its expanded tectopulvinar system, is an ideal species to begin to understand how pathways from the SC influence cortical activity via their projections to the pulvinar nucleus. Previous studies characterized two distinct zones in the tree shrew pulvinar nucleus, a dorsal region (Pd) which receives diffuse convergent input from the SC and projects to the posterior temporal cortex (Tp), and a central region (Pc) which receives specific topographic projections from the SC and projects to the dorsal temporal cortex (Td). We propose to test the idea that separate tectopulvino- cortical pathways are organized to code distinct features of visual movement. To examine this hypothesis we will 1) use in vivo extracellular recordings to compare the activity of Pd and Pc neurons during the presentation a variety of computer generated stimuli designed to test responses to simple and complex visual motion 2) use in vitro whole cell recordings to compare the membrane properties of Pc and Pc neurons and their responses to stimulation of the SC, 3) use tract tracing, immunocytochemistry and electron microscopy to anatomically characterize the connections between the Pd and Pc and temporal cortex, and 4) use in vivo intrinsic optical imaging to examine temporal cortex response pattern changes following deactivation of the SC, Pd or Pc. Given the similarities between the tree shrew and primate visual pathways, the results should further our understanding of human motion processing particularly within the tectopulvinar pathway, and cortical area MT. Therefore, the proposed studies should provide information relevant to the treatment of disorders of motion processing such as dyslexia, schizophrenia, autism and Williams Syndrome. The proposed studies may also provide insight into "blindsight", the ability of some patients to detect visual motion in the absence of visual perception. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): While advances have been made in characterizing the synaptic organization and visual response properties of the pulvinar nucleus, its'exact contributions to the coding of visual signals have remained elusive. The proposed experiments are designed to answer fundamental questions regarding the projection from the pulvinar nucleus to the striate cortex. Using novel combinations of optogenetics, tract tracing and in vitro whole cell recordings, we will determine which cortical cell types are activated by pulvinocortical terminals, and the frequency dependency of pulvinocortical responses. We will then use optogenetics and in vivo intrinsic optical imaging to determine whether inputs from the pulvinar nucleus change cortical receptive field properties. Tree shrews (tupaia belangeri) will be used as the experimental model because the circuitry of tree shrew striate cortex is well defined and similar to that of primates, and in vivo intrinsic optical imaging of the tree shrew striate cortex provides a convenient method to monitor global changes in receptive field properties. The proposed experiments should contribute to our understanding of the construction and modulation of striate cortex response properties, and how the pulvinar nucleus contributes to vision. PUBLIC HEALTH RELEVANCE: The proposed experiments should contribute to our basic scientific understanding of the construction and modulation of V1 response properties relevant to the treatment of disorders such as strabismus and amblyopia. The experiments should also reveal fundamental information regarding the function of the pulvinocortical projections relevant to the treatment of disorders that are known to affect the pulvinar nucleus, such as schizophrenia.

DESCRIPTION (provided by applicant): The superficial layers of the superior colliculus (SC) contain two cell types that both respond to the movement of visual stimuli, but are morphologically and functionally distinct. A projection from the SC to the lateral posterior nucleus (LPN) originates from wide-field vertical (WFV) cells, while a projection from the SC to the dorsal lateral geniculate nucleus (dLGN) originates from narrow-field vertical (NFV) cells. WFV cells have been described as motion detectors based on their responses to small stimuli moving in any direction within a very large receptive field. In contrast, NFV cells may be specialized to code more detailed motion parameters based on their small receptive fields and strong direction selectivity. The parallel WFV and NFV pathways remain segregated in that the tectorecipient dLGN and LPN project differentially to the striate and extrastriate cortex. However, little is known regarding the interaction between the SC, the tectorecipient thalamus, and the cortex. We propose to analyze the circuits that connect these structures in mice by using novel combinations of optogenetics, in vitro whole cell recordings from neuronal populations identified by retrograde tracing techniques, as well as quantitative electron microscopic investigation of synaptic connections. The Aim 1 experiments will use in vitro whole cell recording from identified cortical cell populations and optogenetic activation of terminals tht originate from the tectorecipient dLGN or LPN to determine which cell types are directly innervated and to characterize the electrophysiological properties of these synapses. Electron microscopy will quantify ultrastructural features of these synapses. The Aim 2 experiments will use in vitro whole cell recordings from NFV and WFV cells and optogenetic activation of corticotectal terminals to determine whether these cells receive direct or indirect input from V1 or the lateral extrastriate cortex, and to characterize the electrophysiological properties of thes connections. Electron microscopy will quantify ultrastructural features of corticotectal synapses and the distribution inputs to NFV and WFV cells that do and do not contain gamma amino butyric acid (GABA). As comparisons of parallel geniculocortical pathways have led to insights regarding cortical processing streams, a comparison of WFV and NFV tecto-thalamo-cortical pathways will help us to understand how different aspects of visual motion are utilized by the visual system. In addition, our circuit analysis can help reveal whether corticotectal pathways are organized to enhance segregation, or synthesis, of motion signals

Abstract The flow of visual information from the retina, through the dorsal lateral geniculate nucleus (dLGN) to the cortex, is regulated by behavior. However, the dynamic circuit interactions that occur in the dLGN of awake animals, and their modulation by behavior, have yet to be revealed. The purpose of this proposal is to develop tools to determine how inhibitory circuits of the dLGN (which utilize the neurotransmitter gamma amino butyric acid, GABA) interact in vivo, and how they collectively shape vision in the context of behavior. The premise of this study is based on two key pieces of information: 1) Our previous ultrastructural analyses and in vitro optogenetic experiments suggest that two extrinsic GABAergic inputs to the dLGN, originating from the thalamic reticular nucleus (TRN) and pretectum (PT), serve to suppress or enhance retinogeniculate transmission respectively. 2) Previous studies suggest that the TRN and PT are active during different behavioral states. Thus, we hypothesize that these two sources of inhibition serve to suppress or enhance visual signals in the dLGN during different behavioral states. We propose to test this hypothesis by recording dLGN visual responses in behaving mice while selectively and independently manipulating TRN and/or PT terminals within the dLGN. In head-fixed alert mice, we will record the visual responses of dLGN neurons to computer-generated visual displays while simultaneously recording running speed, eye movements, and pupil diameter. The Aim 1 experiments will test the hypothesis that the PT functions to enhance retinogeniculate transmission immediately following eye movements, to boost cortical activation following visual target acquisition. For this aim, geniculate responses will be recorded during optogenetic silencing or activation of PT terminals, chemogenetic silencing of TRN terminals, or the combined optogenetic/chemogenetic manipulation of PT and TRN terminals. The Aim 2 experiments will test the hypothesis that the TRN dampens retinogeniculate transmission during quiescent states. For this aim, geniculate responses will be recorded during optogenetic silencing or activation of TRN terminals, chemogenetic silencing of PT terminals, or the combined optogenetic/chemogenetic manipulation of TRN and PT terminals. The development of techniques to manipulate circuits in vivo, in addition to our existing anatomical and in vitro experiment strategies, will provide a powerful multipronged approach to deciphering how the individual components of brain circuits are integrated. Once these in vivo methods are perfected, our methodologically-integrated approach can be used to answer a wide variety of outstanding questions regarding thalamic function.

Electron Microscopy Core Summary While methods to image fluorescent molecules have advanced dramatically in recent years, the resolution of electron microscopy remains unsurpassed. In the state of Kentucky, there are numerous research programs that require the resolution that can only be achieved by the interaction of a beam of electrons with a specimen to distinguish ultrastructural features of tissues, cell components, viruses or bacteria. However, due to declining institutional support for electron microscopy facilities, many established Kentucky investigators have utilized facilities and expertise outside the state for their electron microscopic analyses. Furthermore, new Kentucky investigators are hesitant to add electron microscopy to their research programs without accessible training and technical support. We propose to address these gaps in infrastructure and education by establishing a statewide electron microscopy core to facilitate equipment sharing, provide technical support, and offer training.

Administrative Core Summary The primary purpose of the administrative core is to provide sound management of the INBRE-award, on behalf of the network. In addition, this core will provide leadership and direction to enable the network to grow in a purposeful and managed way. The administrative core will provide leadership and oversight of the bioinformatics and research cores, as well as, all other activities, adjusting and balancing the commitments and needs of each, such that the overall network is productive and impactful. The core will be responsible for tracking and evaluation of all network activities, including mentoring and training.

Developmental Research Program (DRPP) - Project Summary The Development Research Project Program provides an array of support programs to develop competitive biomedical research faculty in Kentucky?s colleges and universities. The primary assumption underlying this program is that each Kentucky institution needs a critical mass of independent biomedical research faculty in order to provide the highest quality biomedical research training to undergraduate students. Furthermore, Kentucky research faculty must be provided with the infrastructure to succeed in their research careers in order to provide first-rate research experiences to undergraduate students, which will inspire them to join the biomedical workforce. With the overarching goal of increasing biomedical research capacity in Kentucky, the Developmental Research Project Program aims to address the existing low numbers of independent research faculty, and existing deficits in infrastructure, by enhancing faculty recruitment, promoting the research and career development of junior faculty, and building a sustainable research network. Based upon our past successes, we will focus our efforts on institutions eligible for NIH R15 funding. These institutions are primarily undergraduate institutions (PUIs), so this concentration provides the greatest benefit to undergraduate students. At these institutions, we focus our efforts on developing faculty to increase success rates in obtaining R15 funding. In this way, we have been successful in increasing the numbers of independent research faculty, and building a sustainable research network.

Overall Summary The overarching goal of the Kentucky Institutional Development Award (IDeA) Network of Biomedical Research Excellence (KY-INBRE) is to build capacity for health-related and biomedical research in the Commonwealth of Kentucky. We will build a collaborative research and research-training community throughout the state. Our primary collaborative network is represented by 2 research intensive universities, 5 state-supported and one independent, primarily undergraduate institutions (PUIs). We provide outreach activities in support of many other of our smaller state supported and independent institutions including community technical colleges. Overall, our activities are designed to build on our established multi-disciplinary research network, with a focus on functional genomics, as a means to strengthen research infrastructure of the lead and partner institutions. One aim is to support the development of young research faculty investigators to help them become independently competitive for federal research grants. Another aim of our network is to provide a variety of research training opportunities for our undergraduate and graduate students. These students represent the next generation of the health care and/or biomedical research industries and they need to be mentored to develop appropriate scientific research skills. .

PROJECT SUMMARY / ABSTRACT The concept of parallel pathways that code different aspects of the visual scene has led to many key insights regarding the functional organization of the visual system. Inspired by this concept, the proposed studies focus on parallel visual pathways from the retina to the superior colliculus (SC) through the pulvinar nucleus (PUL). Projections from the SC to the PUL originate from motion-detecting widefield vertical (WFV) cells, and their synaptic organization defines two distinct PUL subdivisions: one that receives ipsilateral topographic WFV projections (?specific?), and one that is innervated by bilateral convergent WFV projections (?diffuse?). These two WFV innervation patterns are correlated with distinct cortical and subcortical connections, as well a variety of histochemical criteria, suggesting that the tectorecipient PUL may be organized into separate visual movement processing streams. However, we currently lack a functional framework that allows us to test this hypothesis and decipher the modular organization of the PUL. We plan to address this gap in knowledge by defining PUL cell types and synaptic inputs in the context of their functional properties. Our guiding hypothesis is that the PUL is composed of two distinct modules that coordinate visual perception with body movements or motivational state to initiate appropriate motor commands. To begin to test this theory, with mice as our animal model, we will use anatomical intersectional viral vector approaches and in vitro whole cell recordings coupled with dual optogenetic activation of cortical and WFV synaptic inputs to define circuit mechanisms that can alter firing properties within each PUL module (Aim 1). We will use in vivo extracellular recordings coupled with optogenetic activation and silencing of synaptic inputs to determine how circuit interactions within each PUL module adjusts receptive field properties (Aim 2). A key innovation of our experiments is the ability to identify PUL neuron subtypes by their unique frequency-dependent responses to optogenetic activation of WFV inputs (?neuron identification via single input dynamics?). This new method will allow us to link detailed in vitro circuit dissection techniques with in vivo recording of visual response properties, providing a framework of PUL function that has thus far been elusive. By comparing two parallel PUL modules, our goal is to understand how visual motion signals are parsed to initiate appropriate behavioral responses.

Project Summary/Abstract Platinum chemotherapeutics are the primary treatment for nonsmall cell lung cancer (NSCLC), but due to the prevalence of resistance to these compounds, the addition of PD-1 immunotherapy has become the standard of care. However, chemotherapy has been shown to impact anti-tumor immunity by inducing PD-1 expression, CD8+ T cell priming, and infiltration leading to immunogenic cell death. There is an urgent need to understand the molecular mechanisms by which platinum-based chemotherapeutics impact the immune response in NSCLC. To analyze the paradoxical effects of platinum compounds on the immune system, a systemically designed and novel bank of platinum compounds with structural similarities to FDA-approved chemotherapeutic will be used. Aim 1) In cellular models of lung cancer, the cell surface expression patterns of T Cell activators will be examined. Both matrix metalloproteases and class I major histocompatibility complex, which play a role in metal sensitivity, will be analyzed before and after exposure to the platinum compounds. We hypothesize that leaving ligand differences of platinum compounds will impact the levels of these factors on the cell surface, correlating to the cell-type-specific variances in platinum toxicity. Aim 2) The impact of the novel platinum compounds on the adenosine dependent pathway through the production of tumor-derived prostaglandin E2 (PGE2) and modulation of the cell surface expression of CD73 will be determined. Our hypothesis is that production of adenosine is stimulated by the indirect effects of chemotherapy on M-MDSCs via the following steps: (a) chemotherapy induces PGE2 production in tumor cells; (b) the released PGE2 upregulates CD73 ecto- 5?-nucleotidase enzyme on the surface of M-MDSCs; (c) CD73 catalyzes the production of adenosine from AMP (derived from ATP released by dying cells); and (d) adenosine inhibits the activation of effector CD8+ T cells within the tumors. The long-term goal of this proposal is to understand the mechanisms that mediate platinum resistance and toxicity in NSCLC, ultimately leading to strategies to block any immunosuppressive effects of chemotherapy. The study is innovative in both the signaling model proposed for the blockade of the immune system and in the design of the platinum compounds that vary only at the leaving ligand.

Project Summary/Abstract Since the World Health Organization declared COVID-19 a pandemic, 31.6 million Americans have contracted the SARS-CoV-2 virus and 566,000 have died. Effective and safe COVID-19 vaccines have been developed, tested, and deployed in record time and 219 million vaccine doses have been administered in the US. Racial and ethnic disparities in vaccine uptake have been noted, but data are not yet available for some other disadvantaged groups. Sexual and Gender Minorities (SGMs) are potentially at risk for vaccine hesitancy due to social and historical factors affecting vaccination in general and healthcare access specifically. There is a critical need to understand vaccine attitudes and behaviors among SGMs. Our long-term goal is to reduce potential health disparities in COVID-19 among SGMs. The overall objective is to develop and deploy a decision aid that supports SGM COVID-19 vaccine decision making. Using survey and focus group methods, and following accepted international standards for decision aid development, we will develop a tailored decision aid to reduce vaccine decisional conflict and improve vaccine acceptance. The project?s specific aims are to: 1. Generate foundational knowledge of COVID-19 vaccine hesitancy among SGMs. We hypothesize that SGMs overall will be somewhat less accepting of the vaccine than the general population and, among SGMs, there will be subgroups with other vaccine hesitancy risk factors (e.g., race, gender minority identity, political affiliation) who have significantly higher rates of vaccine hesitancy. 2. Produce contextualized understanding of intentions to receive the COVID-19 vaccine. We hypothesize the reasons for vaccine acceptance or hesitancy will differ among SGM subgroups. Focus group discussions with diverse samples of SGMs who are vaccine-hesitant will identify knowledge gaps, misconceptions, perceived barriers, negative attitudes such as stigma and mistrust, and preferences for decision support. The primary outcome will be determining decision support needs for SGM subgroups. 3. Create decision aids to reduce decision conflict and improve acceptance of the COVID-19 vaccine. We will generate a decision aid, or multiple versions of a decision aid, to empower the SGM decision maker to compare options for protection against COVID-19, clarify values, and support efficacy for gathering more information, collaborating with a healthcare partner in the decision-making process, and/or obtaining the COVID-19 vaccine. We hypothesize the tailored decision aids will reduce decision conflict and improve vaccine acceptance.

ABSTRACT Perceptual decision-making is a fundamental cognitive ability that is vital to healthy, daily functioning and is impaired in many diseases. Although many brain regions are known to be involved, there is no clear brain-wide model of how perceptual decisions are formed and executed and the underlying circuit mechanisms are still largely unknown. Here, a team of investigators propose a series of experiments that will use behavioral measures, imaging, physiology, circuit dissection, and computational modeling to study how the midbrain superior colliculus (SC) participates in visual decision-making. Specifically, this new team of investigators will probe the contribution of two SC neuronal cell types, wide field vertical (WFV) cells in the visuosensory layers and predorsal bundle (PDB) cells in the motor layers. These experiments will be done in mice and tree shrews, to reveal the underlying circuits and computational principles across species and to lay the foundation for future experiments designed to dissect decision-making circuits in primates. In Aim 1, the investigators will establish and perform psychophysical experiments to assess perceptual decision-making in both species. The behavioral data will be fitted with computational models to arbitrate between different theories of decision-making. In Aim 2, two photon calcium imaging and/or physiological recording will be performed in mice and tree shrews to determine the activity of WFV and PDB neurons during the psychophysical measures established in Aim 1. In addition, WFV and PDB neurons will be silenced optogenetically during the behavioral tasks to reveal their specific roles in decision-making. In Aim 3, the investigators will use intersectional monosynaptic viral tracing techniques, multiplexed peroxidase labeling for confocal and ultrastructural analysis of synaptic connections and and optogenetics-assisted brain slice recording to investigate the intrinsic and extrinsic circuits that link WFV and PDB cells. Together, these experiments will generate novel knowledge of the synapse to circuit mechanisms underlying perceptual decision-making, and provide technical and theoretical foundations for future mechanistic studies of cognitive function in higher mammalian species directly relevant to humans.