Sue A. Aicher - US grants

[unreadable] DESCRIPTION (provided by applicant): Glutamate receptors, particularly AMPA receptors, in the nucleus of the solitary tract (NTS) are the 3rimary mechanism for synaptic transmission between primary afferents and second-order neurons. Thus, an understanding of glutamate receptors in NTS is vital to understanding autonomic reflexes. In our previous studies of AMPA receptors we made a surprising discovery: the number of GluR1 receptor subunits significantly increases in NTS following the development of hypertension. This finding is particularly intriguing since GluR1 is found in dendritic spines, which are widely associated with synaptic plasticity in the brain. Our hypothesis is that structural remodeling in NTS neurons during hypertension leads to functional changes in individual NTS neurons that in turn contribute to alterations in the baroreceptor reflex pathway, such as central baroreflex resetting. These structural changes are found in regions of NTS that contain baroreceptive afferents and second-order neurons, but we do not know which NTS neurons contain GluRI. In the present experiments we will use a multidisciplinary approach to: (1) determine which population(s) of NTS neurons contain GluR1; (2) examine the structural and functional changes in NTS neurons following the development of hypertension; and (3) address the functional relationship between this structural neuronal change and blood pressure. The combined efforts of the Aicher and Andresen laboratories bring together expertise in anatomical and physiological techniques. This collaborative project provides a unique opportunity to directly address structural and physiological questions regarding NTS neuronal function in both the normal and hypertensive states. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Effective treatment of craniofacial pain requires knowledge of the relationships between neural structures, neurotransmitters and receptors that may be therapeutic targets. The physiological basis for these relationships is grounded in the anatomical arrangements between primary afferents, descending projections, interneurons, and efferent projection neurons in the trigeminal dorsal horn. The long-term goal of this work is to understand the anatomical relationship between neuronal elements that mediate or modulate nociception in the trigeminal dorsal horn. The proposed studies use corneal afferents as a nociceptive marker and will examine the relationships between these afferents and other neurons, including interneurons, projection neurons, and descending inputs, in the trigeminal dorsal horn. Studies in Aim 1 will determine if corneal afferents target inhibitory interneurons, trigeminothalamic or trigeminopontine projection neurons; thus defining the basic connectivity of these afferents. Corneal afferents project to two distinct regions of trigeminal dorsal horn, and these regions are thought to mediate functionally distinct components of corneal nociception. Preliminary data demonstrate that the neurotransmitter and transporter content of corneal afferent projections to these two regions are distinct. In Aim 2, differences in the expression of neuronal markers will be examined in corneal afferents, including various putative sensory transduction molecules. Additional studies will determine if select populations of corneal afferents target specific populations of target neurons defined in Aim 1. In Aim 3, the targets of descending medullary projections will be examined to determine if these neurons converge on similar neurons with corneal afferents. Together these studies will provide a more specific understanding of the anatomical relationships between corneal afferents and descending afferent projections, inhibitory interneurons and efferent projection neurons within the trigeminal dorsal horn. These results will provide information about the anatomical substrates for ocular pain and potential mechanisms for both neuronal plasticity and therapeutic targets in the trigeminal dorsal horn. PUBLIC HEALTH RELEVANCE Orofacial pain represents a significant public health concern, afflicting more than 10% of adults, and 50% of elderly adults. Pain arising from corneal injury is on the rise due to the increased number of Americans undergoing refractive eye surgery, including PRK and LASIK (Belmonte, 2004, 2007). Other sources of ocular pain include dry eye, uveitis, and contact lens use. The studies in this proposal are designed to understand the neural pathways involved in transmitting pain from the cornea to the brain.

We propose to establish a Neuroscience Imaging Center at OHSU that will provide state-of-the-art instrumentation for electron, fluorescent, and confocal microscopy as well as expertise in designing and analyzing imaging experiments. The Neuroscience Imaging Center will have 3 cores: 1) a Live-cell Imaging Core, 2) a Confocal Core, and 3) an EM Core. The major instrumentation and expertise necessary for establishing these cores exist already at OHSU. However, the lack of fully staffed cores for these microscopes prevents efficient and reliable use of these instruments by NINDS investigators. The Neuroscience Imaging Center will provide the dedicated staff for these microscopes to enhance and facilitate the research by NINDS investigators at OHSU. An advisory committee, aided by an administrative core, will oversee the operations of the Center, review and optimize the effectiveness of the component cores, and manage the fiscal aspects of the Center. The three cores will function together as a truly integrated facility from experimental design, to performance, to data analysis. Instrumentation resources will be located on both the West and Marquam Hill campuses, thereby serving the entire OHSU neuroscience community. Drs. Sue Aicher and Gary Banker will serve as co-Directors of the Center;each Director brings unique scientific and technical qualifications and experience in managing shared facilities. The Center Directors will provide front-end consultation with the investigators, refining experimental questions and formulating optimal technical approaches for individual experiments. Three Core Managers and technical staff will operate the major instrumentation, train new users, and provide full service experiments for some investigators. Training opportunities will be available to serve principal investigators, postdoctoral fellows, and graduate students. Neuroscience is the centerpiece of research at OHSU;the Imaging Center will bring cutting-edge imaging to the 23 NINDS-funded research programs identified in this proposal, as well as our NINDS-funded trainees. The Neuroscience Imaging Center will catalyze focused interactions within our entire neuroscience community.

Project Summary The Oregon Health & Science University (OHSU) Neuroscience Imaging Center will provide expertise in designing, conducting, and analyzing experiments on state-of-the-art instrumentation for light and electron microscopy. The Neuroscience Imaging Center has two scientific Cores: Core 1 - Analytical Fluorescence Imaging and Core 2 - Ultrastructure and Single Particle Microscopy. The instruments for these Cores are in place and the technical capacities have been expanded tremendously over the past six years to include not only confocal microscopy and transmission electron microscopy, but also in vivo two-photon microscopy, super-resolution microscopy, EM tomography and single particle analyses for determining molecular structures. All instruments are located in university-wide service centers that are supported by the office of the Vice President for Research. The Neuroscience Imaging Center serves NINDS-funded OHSU researchers, as well as other NIH-funded investigators engaged in neuroscience research. We currently have 21 NINDS- funded investigators who have submitted qualifying projects for the current proposal and have microscopy and imaging needs for their current research. Other OHSU neuroscientists also receive support from Core staff. User fees on shared instruments are partially supported by the P30 grant, with greater support for NINDS than non-NINDS investigators; user fee support is regularly reviewed by the Steering Committee. Microscopy core facilities are located on the Marquam Hill campus and at the adjacent Waterfront Campus (see Resources), and all P30-supported investigators will have open access to all of these resources. The OHSU Neuroscience Imaging Center grant provides salary support for Core staff, who are local, service-oriented technology experts. The Neuroscience Imaging Center Director Dr. Sue Aicher and Associate Director Dr. Catherine Morgans have extensive scientific and technical expertise and will provide front-end consultation with investigators, helping to direct each investigator to the appropriate resources to address their research goals. The Steering Committee provides input to the Center Director with regard to Core strategic and operational issues, as well as project prioritization and Core access plans. We provide different levels of support based on the needs of the investigators. Investigators with extensive imaging expertise primarily require access to state- of-the-art equipment and occasional consultation with staff. Other investigators need more in-depth support, and full service support is often provided for electron microscopy studies. The P30 staff will provide expert support at the appropriate level to meet the needs of our participating investigators. The Neuroscience Imaging Center has catalyzed interactions among NINDS researchers as well as other neuroscientist at OHSU, provided synergy for investments in advanced microscopy, and significantly enhanced scientific progress for our neuroscience community.

Project Summary The Oregon Health & Science University (OHSU) Neuroscience Imaging Center will provide expertise in designing, conducting, and analyzing experiments on state-of-the-art instrumentation for light and electron microscopy. The Neuroscience Imaging Center has two scientific Cores: Core 1 - Analytical Fluorescence Imaging and Core 2 - Ultrastructure and Single Particle Microscopy. The instruments for these Cores are in place and the technical capacities have been expanded tremendously over the past six years to include not only confocal microscopy and transmission electron microscopy, but also in vivo two-photon microscopy, super-resolution microscopy, EM tomography and single particle analyses for determining molecular structures. All instruments are located in university-wide service centers that are supported by the office of the Vice President for Research. The Neuroscience Imaging Center serves NINDS-funded OHSU researchers, as well as other NIH-funded investigators engaged in neuroscience research. We currently have 21 NINDS- funded investigators who have submitted qualifying projects for the current proposal and have microscopy and imaging needs for their current research. Other OHSU neuroscientists also receive support from Core staff. User fees on shared instruments are partially supported by the P30 grant, with greater support for NINDS than non-NINDS investigators; user fee support is regularly reviewed by the Steering Committee. Microscopy core facilities are located on the Marquam Hill campus and at the adjacent Waterfront Campus (see Resources), and all P30-supported investigators will have open access to all of these resources. The OHSU Neuroscience Imaging Center grant provides salary support for Core staff, who are local, service-oriented technology experts. The Neuroscience Imaging Center Director Dr. Sue Aicher and Associate Director Dr. Catherine Morgans have extensive scientific and technical expertise and will provide front-end consultation with investigators, helping to direct each investigator to the appropriate resources to address their research goals. The Steering Committee provides input to the Center Director with regard to Core strategic and operational issues, as well as project prioritization and Core access plans. We provide different levels of support based on the needs of the investigators. Investigators with extensive imaging expertise primarily require access to state- of-the-art equipment and occasional consultation with staff. Other investigators need more in-depth support, and full service support is often provided for electron microscopy studies. The P30 staff will provide expert support at the appropriate level to meet the needs of our participating investigators. The Neuroscience Imaging Center has catalyzed interactions among NINDS researchers as well as other neuroscientist at OHSU, provided synergy for investments in advanced microscopy, and significantly enhanced scientific progress for our neuroscience community.

Project Summary The Oregon Health & Science University (OHSU) Neuroscience Imaging Center will provide expertise in designing, conducting, and analyzing experiments on state-of-the-art instrumentation for light and electron microscopy. The Neuroscience Imaging Center has two scientific Cores: Core 1 - Analytical Fluorescence Imaging and Core 2 - Ultrastructure and Single Particle Microscopy. The instruments for these Cores are in place and the technical capacities have been expanded tremendously over the past six years to include not only confocal microscopy and transmission electron microscopy, but also in vivo two-photon microscopy, super-resolution microscopy, EM tomography and single particle analyses for determining molecular structures. All instruments are located in university-wide service centers that are supported by the office of the Vice President for Research. The Neuroscience Imaging Center serves NINDS-funded OHSU researchers, as well as other NIH-funded investigators engaged in neuroscience research. We currently have 21 NINDS- funded investigators who have submitted qualifying projects for the current proposal and have microscopy and imaging needs for their current research. Other OHSU neuroscientists also receive support from Core staff. User fees on shared instruments are partially supported by the P30 grant, with greater support for NINDS than non-NINDS investigators; user fee support is regularly reviewed by the Steering Committee. Microscopy core facilities are located on the Marquam Hill campus and at the adjacent Waterfront Campus (see Resources), and all P30-supported investigators will have open access to all of these resources. The OHSU Neuroscience Imaging Center grant provides salary support for Core staff, who are local, service-oriented technology experts. The Neuroscience Imaging Center Director Dr. Sue Aicher and Associate Director Dr. Catherine Morgans have extensive scientific and technical expertise and will provide front-end consultation with investigators, helping to direct each investigator to the appropriate resources to address their research goals. The Steering Committee provides input to the Center Director with regard to Core strategic and operational issues, as well as project prioritization and Core access plans. We provide different levels of support based on the needs of the investigators. Investigators with extensive imaging expertise primarily require access to state- of-the-art equipment and occasional consultation with staff. Other investigators need more in-depth support, and full service support is often provided for electron microscopy studies. The P30 staff will provide expert support at the appropriate level to meet the needs of our participating investigators. The Neuroscience Imaging Center has catalyzed interactions among NINDS researchers as well as other neuroscientist at OHSU, provided synergy for investments in advanced microscopy, and significantly enhanced scientific progress for our neuroscience community.

PROJECT SUMMARY Photorefractive keratectomy (PRK) and laser-assisted in situ keratomileusis (LASIK) are surgical procedures used to correct refractive errors, with approximately 600,000 surgeries performed in the United States each year. A proportion (10 - 20%) of individuals develop persistent ocular pain after refractive surgery, which is a source of tremendous morbidity, limiting the ability to work, impacting mental health, and leading to suicidal ideation in severe cases. We hypothesize that proteins in the tear fluid modulate corneal nerve function, increasing the risk of and contributing to persistent pain after refractive surgery. As such, our studies will use proteomic analysis of tear fluid after surgery to identify diagnostic biomarkers for persistent ocular pain. We will also examine tear proteins prior to surgery in an effort to identify prognostic biomarkers that predict which individuals are likely to develop persistent pain after surgery. R33 Phase studies will use an alternative proteomics platform for analytical validation, and an additional new patient population for clinical validation. This knowledge can lead to better preventative and therapeutic algorithms to combat this debilitating condition and thus improve quality of life and decrease patient suffering.

SUMMARY Electrical synapses, also known as gap junctions, occur frequently in all nervous systems, including the human brain. They are composed of connexins, arranged to form intercellular channels between adjacent, coupled cells. Connexin36 (Cx36) is the predominant connexin in the CNS. In many brain and retinal circuits, gap junctions provide direct and specific connections between cells. In addition, electrical synapses mediate network properties such as signal averaging, noise reduction and synchronization. However, because of their small size, gap junctions are not visible in large-scale serial EM data sets. For these reasons, gap junctions tend to be under-reported or simply ignored. The objective of this proposal is to develop a combined approach to image gap junction connectivity in EM datasets and, in addition, to estimate the size, strength, and plasticity of gap junctions. We will study regions of the retina that contain gap junctions of dramatically different sizes and shapes, to allow us to correlate structure and function. Aim 1 will use high-resolution confocal microscopy to determine connexon number at large and small gap junctions. Analyses will determine the number of connexons per gap junction. These methods will provide a general-purpose tool to determine the size of gap junctions for use in all brain regions. Aim 2 will use 3D-EM imaging to allow unambiguous identification of gap junctions in FIB-SEM images, which will follow with first-ever immunogold quantification of a membrane-bound protein in 3D-EM structures. These studies will allow high-resolution quantification of gap junctions and proteins in identified neurons. Aim 3 will use electrophysiological measures to determine coupling conductance and then develop models to calculate the maximal potential coupling conductance from the morphological data by multiplying the number of channels/gap junction [Specific Aim 1] times the connectivity (the number of gap junctions between coupled cells) [Specific Aim 2], times the unitary conductance of Cx36. Using paired recordings, we will obtain direct physiological measures of the junctional conductance between coupled cells. Then, by comparison with the potential maximum calculated from the morphological data, we can calculate the open channel probability and place realistic limits on the operating range. These are the fundamental properties required to understand the function of gap junctions in neuronal microcircuits. This program is an exact match for one of the listed areas, ?Tools to identify gap junctions and characterize electrical synapses? in the Funding Opportunity Announcement, RFA-MH-20-135.