Ellen A. Lumpkin, Phd - US grants

DESCRIPTION (provided by applicant): The long-term goal of this research is to elucidate the molecular basis of mechanotransduction by mammalian somatosensory receptor cells using the cutaneous Merkel cell-neurite complex as a model. Somatosensory mechanoreceptors mediate the senses of touch, pain and proprioception. The importance of these senses to human health is underscored by diseases that cause peripheral neuropathy such as rheumatoid arthritis, diabetes and acquired immunodeficiency syndrome. Because patients with peripheral neuropathy cannot feel injuries, even minor insults can lead to irreversible tissue damage and chronic pain. Although Merkel cells are conserved throughout vertebrate evolution, their function is unknown. Merkel cells have been proposed to be mechanoreceptors that transduce pressure and convey this information to the nervous system through synaptic transmission. Alternatively, Merkel cells may influence the activity of mechanosensory neurons through the release of neuromodulators. Finally, Merkel cells may affect the development of touch responsiveness, either by influencing the innervation patterns of sensory neurons or by directing neighboring epidermal cells to form specialized skin structures that are highly sensitive to touch. The objective of this application is to distinguish between these proposed functions by developing molecular and in vitro tools. First, we will use mouse DNA microarrays to identify gene-expression profiles of Merkel cells during and after maturation of the sense of cutaneous touch. Second, we will develop methods for studying ion channels and neurotransmitter release in dissociated Merkel cells. These in vitro methods will then be used to examine the roles of Merkel-cell-enriched molecules in signaling. By pinpointing molecules that may be essential for Merkel-cell signaling, these studies lay the groundwork for future investigation of the role of this epidermal cell in touch reception, and of the signals that initiate our perception of touch and pain. The proposed Specific Aims are: 1. Identify transcripts that are enriched in Merkel cells 2. Define the ionic conductances of dissociated Merkel cells 3. Characterize neurotransmitter release from Merkel cells in vitro

DESCRIPTION (provided by applicant): This application is for funds to purchase a BD Biosciences Special Order LSRII quadruple-laser FACS analyzer that will be situated in the new Cytometry and Cell Sorting Core at Baylor College of Medicine (BCM). This instrument has been selected to serve the purposes of over 16 investigators involved in over 18 NIH- funded projects. This instrument has been designed to serve two key purposes in our community. First, many BCM investigators require the capacity to analyze on multiple fluorescent markers concomitantly with multiple cell surface markers. Our four-laser instrument will allow analysis based on multiple parameters simultaneously such as mCherry, cyan fluorescent protein, yellow fluorescent protein, and allophycocyanin (APC) or sytox red. It will have a 561-nm solid-state laser, allowing detection of red fluorescent protein derivatives such as mCherry and increasing the sensitivity of detecting commonly used probes such as phycoerythrin. It will also be equipped with a 405-nm solid-state laser enabling the use of violet sensitive probes including cyan fluorescent protein and the fluorochrome Pacific Blue. Additionally, a 640-nm laser will allow detection of fluorochromes such as APC and its conjugates as well as the viability dye sytox red, which will give added power and flexibility to all investigators needing multiple easy-to-use parameters for analysis. Finally, the instrument will be equipped with a 488-nm solid-state laser enabling detection of standard fluorophores including green fluorescent protein, yellow fluorescent protein and fluorescein. The second key purpose for this flow analyzer is small-particle detection to study bacteria and yeast, which is critical for four groups of investigators at BCM. Thus, the instrument will include a forward scatter photo-multiplier tube detector enabling small particle detection and analysis, directly enhancing the research using bacteria and yeast. This instrument will afford maximum capabilities as well as maximum flexibility to best serve the evolving needs of the diverse BCM investigators and Texas Medical Center research community. This instrument is essential for the broad base of health-related research at BCM, including projects aimed at developing new therapies in regenerative medicine, cancer, HIV/AIDS, immunology and aging, which will contribute to the overall wellness of our society. Public Health Relevance: Our research at the Baylor College of Medicine requiring a four-laser LSRII flow cytometer will specifically study processes within cells allowing us to better understand stem cell therapies, aging processes, cancer biology and cardiovascular development. The requested LSRII flow cytometer is critical for determining the complexity and differences between individual cells, which is essential for our research projects aimed at developing new treatments or cures for serious human illnesses including cancer, heart abnormalities, stroke and HIV/AIDS.

? DESCRIPTION (provided by applicant): The sense of touch is vital for our survival, as it is essential for purposes as simple as picking up a fork and as complex as bonding with a newborn. Despite its importance for accomplishing basic tasks, the molecular and cellular mechanisms that convert a force on the skin into a neural signal are poorly understood. The long term goal of this research is to elucidate how receptors in the skin convert a touch stimulus into a neural signal to inform our brains about the physical world. We use Merkel cell-neurite complexes, which are conserved discriminative touch receptors, as a model to study these processes. Merkel cells are epithelial derived target cells that contact the endings of sensory neurons that send information to the central nervous system. Stimulating these Merkel cell-neurite complexes generates slowly adapting type I responses, which are important for detecting object features such as edges and curvature. Our previous studies revealed that Merkel cells directly sense mechanical force and convey this information to the underlying sensory neurons. The molecules that govern this process are completely unknown. Numerous molecules involved in synaptic transmission and neurotransmitters have been identified in Merkel cells, yet attempts to identify signaling mechanisms in vivo have yielded contradictory results. Thus, the objective of this research is to identify mechanisms by which Merkel cells convey tactile information to the nervous system. We will accomplish this goal by using an innovative combination of transgenic mice, optogenetics, in vitro and ex vivo physiology, and behavioral studies. Our central hypothesis is that Merkel cells release neurotransmitters at TeNT-sensitive SNARE-dependent chemical synapses to excite firing in tactile afferents. In Aim 1, we will determine whether Merkel cells employ TeNT-sensitive SNARE-mediated vesicular release. Aim 2 will identify the neurotransmitter or neuromodulatory pathways that excite Merkel-cell afferent neurons. These studies will yield fundamental insights into peripheral tactile mechanisms, which is essential to advance basic knowledge of principles that govern encoding of somatic senses.

DESCRIPTION (provided by applicant): The long-term goal of this research is to determine how mammalian touch receptors transduce forces into neural signals that inform the brain about objects in our dynamic environment. The sense of touch is essential for behaviors that range from avoiding bodily harm to vital social interactions such as child rearing. The touch receptors that innervate the skin are likewise diverse in their peripheral morphologies and physiological outputs. Previous studies demonstrate that different classes of touch receptors produce distinctive firing patterns that encode spatial and temporal features of objects. Despite past progress, the principles that govern neural output in mammalian touch receptors have not been defined. The objective of this application is to elucidate cellular and systems-level mechanisms that generate neural signals in mouse Merkel cell-neurite complexes, which we use as a model for molecular, physiological and computational studies. These complexes mediate slowly adapting type I (SAI) touch responses, which resolve fine spatial details, such as Braille patterns. Our ability to extract edges and object curvature with high speed and fidelity may relate directly to the SAI afferent's distinctive biphasic firing pattern. The SAI afferent's morphology is also unique among touch receptors because it is synaptically coupled to sensory receptor cells. Each SAI afferent has a branching arbor that contacts ~10-40 Merkel cells. The evolutionary maxim 'form follows function'leads to our central hypothesis that the SAI afferent's unique architecture is fundamental to its distinctive firing properties. This new collaborative project will test this hypothesis by combining computational models, microscopy and neurophysiology. We will build novel computational models using solid mechanics, differential equations and statistics to define the key principles that dictate biphasic SAI firing patterns. To inform the modeling, we will elucidate the three dimensional architecture of mouse SAI afferents, including the quantity and arrangement of Merkel cells and action potential initiation zones. The resulting models will make specific predictions about biological mechanisms that underlie touch-evoked responses in mammals. These predictions will then be experimentally tested with neurophysiological recordings from transgenic mice that allow direct visualization of Merkel cells in receptive fields. The intellectual merit of the proposed research lies in our means of joining computational and experimental techniques to determine how touch-receptor anatomy governs physiology. The power of computation allows us to evaluate thousands of possibilities that would be virtually impossible to empirically test one by one. The power of experimental observation allows us to construct realistic models by visualizing specific anatomical structures and molecules, as well as by measuring neuronal outputs. This strategy fits into an emerging paradigm of biological exploration - that of building predictive models to first explore questions in a modeling space and to subsequently test predictions in empirical space. This project is a new venture between researchers in systems engineering and neurobiology whose careers are dedicated to understanding touch. This research proposal describes a new collaborative project that will benefit from infrastructure developed through our recent study of skin mechanics, which resulted in peer-review manuscripts and conference papers [1, 2, 3, 4]. The broader impacts resulting from the proposed research will be to advance the understanding of force transduction mechanisms in biological systems. This project will support teaching and graduate student training in systems engineering, neuroscience and physiology. The biological principles elucidated in this work may further the understanding of neural signaling in other sensory modalities including pain. We expect the models to be critical for engineering artificial touch sensors that can interface with the human nervous system to restore touch sensitivity (e.g., in burn victims and amputees), as well as for applications in human-robotic manipulation in medicine. We expect the experimental results to impact researchers in fields of sensor design, tissue modeling, neurobiology, psychophysics, haptics, and dermatology. Results will be disseminated in appropriate peer-reviewed journals and conference presentations.

The goal of the Flow Cytometry Shared Resource to provide users with cost-effective instrumentation, expertise and training for cell sorting and analysis. This technology continues to develop at a rapid pace, especially with the advent of novel fluorescent reporters, increased computational capacity and more cost-effective optical equipment. To meet our members' increasing demands for state-of-the-art flow cytometry, the DLDCC and BCM administration collaborated to create an entirely new flow cytometry facility in 2007. Renovation, operating costs and instrumentation has been supported by $1.7 million in BCM institutional funds and >$600,000 in DLDCC funds. The revamped Facility is housed in newly renovated, centrally located space, which is available to trained users 24 h a day via key-card access. State-of-the-art instrumentation, all of which has been purchased in the last three years, includes two fully loaded florescence-activated cell sorters, three flow analyzers and a magnetic cell separator. The Resource is directed by Dr, Ellen A. Lumpkin, who has over nine years of experience in flow cytometry, and Mr. Joel Sederstrom, who was recruited from the Univ. of Minnesota's Cancer Center Flow Cytometry Core in a national search. To ensure optimal use of services, the Resource provides consultations, training and protocols for sample preparation, flow analysis and cell sorting. The Resource is also staffed with two full-time experienced flow cytometrists who perform operator-assisted cytometry, and assist users with data analysis. With the Resource's improved services and capacity, FACS sorting has increased by >500% and FACS analysis has increased 160% among Cancer Center members. At present, the Resource operates near 100% of its capacity, with 78% of usage occupied by 65 Cancer Center investigators whose membership spans all Scientific Programs. Future plans include further expanding services by recruiting an additional cytometrist and by including a second site at our affiliated institution, Texas Children's Hospital Cancer Center.

DESCRIPTION (provided by applicant): The sense of touch is an integral component of countless essential behaviors such as feeding, social exchange and avoiding bodily harm. In mammals, different tactile qualities are encoded by touch receptors with distinct physiological properties and morphological end organs;however, the cellular mechanisms underlying this diversity is unknown. The long-term goal of this research is to determine how mammalian Merkel cell-neurite complexes transduce touch stimuli into neural signals that inform the brain about objects in our dynamic environment. The objective of this application is to determine whether epidermal Merkel cells play an excitatory role in cutaneous touch reception. In complex with myelinated cutaneous afferents, they form gentle-touch receptors that mediate slowly adapting type I (SAI) responses. This project is highly relevant to human health because 1) SAI responses in the skin are thought to underlie high tactile acuity in humans and other mammals that rely on discriminative touch for recognizing and manipulating objects;and 2) Merkel cells are one of only four fundamental cell types in the mammalian epidermis and yet their biological function in the skin remains unknown 135 years after their discovery. Based on morphology, Merkel cells have long been proposed to be mechanosensory cells, analogous to inner-ear hair cells. Alternatively, Merkel cells might be accessory cells that shape sensory output of mechanosensitive SAI afferents. This exploratory project will use innovative technologies to directly distinguish between these two models. This application's central hypothesis is that Merkel cells release excitatory neurotransmitters to evoke action potentials in SAI afferents. To test this hypothesis, Merkel cells must be selectively excited without activating intrinsic mechanosensory mechanisms that might be present in SAI afferents. Since Merkel cells and SAI afferents are tightly juxtaposed in the skin, touch stimuli cannot be used to independently activate each cell type in situ. To break this barrier, the approach will employ a combination of optogenetics, mouse models and ex vivo skin-nerve recordings. Because mouse Merkel cells reside in the transparent epidermis, the light-gated ion channel channelrhodopsin-2 (ChR2) provides an ideal tool to excite Merkel cells in the intact skin. Aims are to 1) achieve selective and robust expression of ChR2 in Merkel cells in vivo, and 2) determine whether exciting Merkel cells in the absence of touch stimuli drives action potential firing in SAI afferents. The project is innovative because it represents first use of a light-activated protein to dissect the function of any skin cell type or to address mechanisms of touch reception. Furthermore, a novel Rosa26ChR2 knock-in mouse model has been generated that, when validated, can be broadly used by the scientific community. PUBLIC HEALTH RELEVANCE: Touch is a fundamental sense necessary for navigating our physical world, yet almost nothing is known about the way that cells in the skin initiate the sense of touch. Merkel cell-neurite complexes are gentle-touch receptors that convey tactile information regarding an object's texture, shape and curvature, and these studies will determine the specific function of epidermal Merkel cells in sensory coding. This project will resolve a key open question in the field of somatosensory coding, which is essential to understand the sense of touch in both healthy skin and pathophysiological pain states.

DESCRIPTION (provided by applicant): The long-term goal of this research is to determine how mammalian touch receptors transduce forces into neural signals that inform the brain about objects in our dynamic environment. The sense of touch is essential for behaviors that range from avoiding bodily harm to vital social interactions such as child rearing. The touch receptors that innervate the skin are likewise diverse in their peripheral morphologies and physiological outputs. Previous studies demonstrate that different classes of touch receptors produce distinctive firing patterns that encode spatial and temporal features of objects. Despite past progress, the principles that govern neural output in mammalian touch receptors have not been defined. The objective of this application is to elucidate cellular and systems-level mechanisms that generate neural signals in mouse Merkel cell-neurite complexes, which we use as a model for molecular, physiological and computational studies. These complexes mediate slowly adapting type I (SAI) touch responses, which resolve fine spatial details, such as Braille patterns. Our ability to extract edges and object curvature with high speed and fidelity may relate directly to the SAI afferent's distinctive biphasic firing pattern. The SAI afferent's morphology is also unique among touch receptors because it is synaptically coupled to sensory receptor cells. Each SAI afferent has a branching arbor that contacts ~10-40 Merkel cells. The evolutionary maxim 'form follows function' leads to our central hypothesis that the SAI afferent's unique architecture is fundamental to its distinctive firing properties. This new collaborative project will test this hypothesis by combining computational models, microscopy and neurophysiology. We will build novel computational models using solid mechanics, differential equations and statistics to define the key principles that dictate biphasic SAI firing patterns. To inform the modeling, we will elucidate the three dimensional architecture of mouse SAI afferents, including the quantity and arrangement of Merkel cells and action potential initiation zones. The resulting models will make specific predictions about biological mechanisms that underlie touch-evoked responses in mammals. These predictions will then be experimentally tested with neurophysiological recordings from transgenic mice that allow direct visualization of Merkel cells in receptive fields. The intellectual merit of the proposed research lies in our means of joining computational and experimental techniques to determine how touch-receptor anatomy governs physiology. The power of computation allows us to evaluate thousands of possibilities that would be virtually impossible to empirically test one by one. The power of experimental observation allows us to construct realistic models by visualizing specific anatomical structures and molecules, as well as by measuring neuronal outputs. This strategy fits into an emerging paradigm of biological exploration - that of building predictive models to first explore questions in a modeling space and to subsequently test predictions in empirical space. This project is a new venture between researchers in systems engineering and neurobiology whose careers are dedicated to understanding touch. This research proposal describes a new collaborative project that will benefit from infrastructure developed through our recent study of skin mechanics, which resulted in peer-review manuscripts and conference papers [1, 2, 3, 4]. The broader impacts resulting from the proposed research will be to advance the understanding of force transduction mechanisms in biological systems. This project will support teaching and graduate student training in systems engineering, neuroscience and physiology. The biological principles elucidated in this work may further the understanding of neural signaling in other sensory modalities including pain. We expect the models to be critical for engineering artificial touch sensors that can interface with the human nervous system to restore touch sensitivity (e.g., in burn victims and amputees), as well as for applications in human-robotic manipulation in medicine. We expect the experimental results to impact researchers in fields of sensor design, tissue modeling, neurobiology, psychophysics, haptics, and dermatology. Results will be disseminated in appropriate peer-reviewed journals and conference presentations.

DESCRIPTION (provided by applicant): Touch is integral to essential behaviors such as feeding, social bonding and avoiding bodily harm. In mammals, touch is encoded by sensory receptors embedded in the skin. Mammalian skin structure and mechanical properties are dynamic, changing in response to numerous physiological and external conditions, including nutrition, body weight, aging and exposure to environmental factors, such as UV irradiation. Little is known about how these physiologic changes alter neuronal signaling from touch receptors. The objective of this application is to elucidate peripheral mechanisms that govern the firing properties of tactile afferents during normal physiological target-organ changes. The project focuses on mouse slowly adapting type I (SAI) afferents as a model system with unparalleled accessibility for computational and experimental studies. Merkel cells in contact with myelinated cutaneous afferents form gentle-touch receptors that mediate SAI responses. This project is highly relevant to human health because 1) SAI responses in the skin underlie high tactile acuity in humans but little is known about how physiological skin remodeling alters their signaling; and 2) understanding mechanisms of normal neuronal remodeling could identify targets for treating pathological or age-related changes in touch sensitivity. Anatomical studies have shown that skin innervation density changes during normal hair growth in mice. This application will address key open questions: 1) what are the mechanisms that govern innervation changes during hair-follicle cycling, and 2) do changes in innervation lead to altered sensory signaling? The central hypothesis is that structural plasticity of tactile afferents govern touch-evoked firing properties during normal skin remodeling. The hypothesis will be tested with an innovative multidisciplinary approach combining experimental techniques, including neurophysiology, 3D microscopy, quantitative morphometry, tissue biomechanics and novel mouse models, with computational tools such as novel network models of neuronal dynamics, differential equations and solid mechanics. Aims are to 1) define temporal dynamics and cellular mechanisms of neuronal remodeling during skin renewal, 2) analyze the functional consequences of neuronal remodeling on mechanical encoding, and 3) identify the target cell type and candidate molecular cues that drive neuronal remodeling. This project is conceptually innovative because it tackles a novel question in basic neurobiology that is central to the encoding of touch stimuli. Technically innovation lies in its unique, interdisciplinary approaches to combine experimental biology with computational studies to answer these fundamental questions. By identifying mechanisms that govern the reliability of touch-evoked signals in healthy skin, these studies will set the stage to determine how these mechanisms fail in aging and pathophysiological states.

The sense of touch enables numerous behaviors fundamental to human existence, allowing individuals to eat, communicate and survive. With this sense, people can discern a surface's roughness, stickiness and vibration, among other attributes. A particularly critical dimension is object compliance. Interactions with compliant objects are pervasive in the world, whether with muscle and tissue, the hands of others, fruits and vegetables, or manufactured elastics. Despite prior psychophysical efforts to identify salient cues between the skin and complaint objects, very little is understood about the underlying neural codes. In particular, how can a diverse population of mechanosensory neurons encode perceptible differences in compliance ? given a rich diversity of stimulus-response transformations, conduction velocities, receptive field characteristics, densities and arrangements? This application's central hypothesis is that cues signaling compliance are reflected in the population response of different types of cutaneous mechanoreceptors, in time-dependent output based on spatial positioning. The hypothesis will be addressed by: i) establishing a new computational paradigm for the in silica generation and validation of population codes empowered by calcium imaging of populations of neurons combined with single-unit neurophysiology and ii) using this novel, intermediate observation point to understand how distinct, naturalistic properties of compliant stimuli are encoded in the periphery. This effort focuses on mouse somatosensory afferents innervating glabrous skin as a tractable mammalian system for computational, experimental and genetic studies.