William Guido - US grants

DESCRIPTION (provided by applicant): Much of our present understanding regarding the activity-dependent refinement of sensory connections is based on work done in the developing retinogeniculate pathway. Despite the overwhelming evidence underscoring the role of activity in shaping the refinement of retinogeniculate connections, the cellular and molecular mechanisms underlying these processes remain a topic of intense inquiry. We have developed a rodent model of visual system development in which we characterized the structural and functional changes occurring in the lateral geniculate nucleus (LGN) during early postnatal life. Our experiments reveal that the changes in retinogeniculate axon patterning and connectivity are linked to Hebbian-like modifications in synaptic strength and the activation of L-type Ca2+ channels. However, the role of L-type Ca2+ channels and their expression during retinogeniculate development remain largely unexplored. The major goals of this project is to investigate the nature of L-type Ca2+ activity during early postnatal life and establish a direct link between such activity and the remodeling of retinogeniculate connections. We shall use electrophysiological, anatomical, and biochemical techniques to delineate the nature of L-type Ca2+ channel expression and its role in retinogeniculate development. We shall take advantage of transgenic mice lines in which L-type Ca2+ activity and channel expression has been severely attenuated by the targeted deletion of either the beta2 or beta3 subunit of the Ca2+ channel. These mice offer a unique opportunity to relate L-type Ca2+ function with synapse stabilization and activity dependent remodeling in LGN.

neurotoxicology; fetal alcohol syndrome; embryo /fetus toxicology; ethanol; developmental neurobiology; alcoholic beverage consumption; NMDA receptors; gestational age; vision; retina; brain electrical activity; lateral geniculate body; cell population study; pregnancy; GABA receptor; voltage gated channel; female; electrophysiology; laboratory rat; electrodes;

DESCRIPTION (provided by applicant): Much is known about how inputs from sources other than the retina affect the thalamic relay of retinal signals in route to the visual cortex. They arse from a variety of subcortical and cortical sources, comprise the bulk of synapses onto thalamic relay cells and act as modulators to affect the gain of retinogeniculate signal transmission. The superior collliculus (SC), a primary retino-recipient structure, also sends a prominent projection to the dorsal lateral geniculate nucleus (dLGN), the first order nucleus that conveys retinal signals to visual cortex. However, whether SC input acts as a driver to help shape the receptive field structure of dLGN relay neurons or as a modulator to dampen or amplify the flow of thalamic information remains untested. To understand how information arising from the SC influences dLGN function and how particular SC cell types contribute to this, we shall make use of mouse transgenics to visualize or target cell types and projections of the tectogeniculate pathway, optogenetics to photo-activate SC inputs to evoke postsynaptic activity in dLGN cells, and in vitro and in vivo recording techniques to assess whether SC inputs behave as drivers or modulators of dLGN activity. These combined approaches will allow us to selectively control the neuronal activity of the tecto-geniculate pathway and shed light on how interactions between two retino- recipient subcortical structures affect the processing and transfer of visual information.

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.

Project Summary/Abstract The thalamic reticular nucleus (TRN), is a shell like structure that surrounds the dorsal thalamus and serves as a key inhibitory interface for the bidirectional signaling between thalamus and the neocortex. Together with inputs from thalamus, cortex, and cholinergic nuclei of brainstem and basal forebrain, the TRN regulates many aspects of sensory, motor, and cognitive processing. When the connections between these structures are disrupted by disease, degeneration, or trauma, they have devastating consequences. In fact, many adult and childhood neurological disorders have at their core, a disturbance in TRN signaling and circuitry. Despite its key role in thalamocortical function, remarkably little is known about how reticular circuitry emerges during development and becomes operational. To address this substantive gap in knowledge we developed a robust mouse model as an experimental platform to visualize, manipulate, and dissect emergent and developing reticular circuitry. We plan to conduct anatomical, electrophysiological, and optogenetic experiments in genetically modified mice that allow for the visualization and experimental manipulation of specific cell types arising from the TRN, first-order thalamic sensory nuclei, layer VI of cortex, and cholinergic nuclei of brainstem and basal forebrain. The goals of this proposal focus on three unanswered questions about TRN development. First, how are the sensory sectors of TRN established; do inputs from primary sensory thalamic nuclei and corresponding regions of cortex innervate TRN diffusely and then segregate to form modality specific domains? Second, what is the sequence and pattern of driver and modulator innervation of TRN; do driver-like inputs from sensory thalamic nuclei such as the dorsal lateral geniculate nucleus, arrive prior to modulatory input from cortex, or brainstem and basal forebrain? Third, how and when do feed-forward and feedback circuits linking TRN to thalamus and cortex emerge during development to control thalamocortical signaling? Finally, for each of these questions, we plan to take a loss of function approach to assess whether the absence of sensory input (vision) affects the development, form, and function of reticular circuitry. These studies will provide valuable information about the organizing principles that guide the emergence of reticular circuitry in the neonatal brain, and perhaps reveal a new understanding into childhood disorders that result from abnormal patterns of connectivity.

Project Summary/Abstract This application is a resubmission for a T35 summer medical student training program in vision research with two Specific Aims: 1) To provide hands-on training in basic and clinical vision research to medical students in a structured mentored environment and 2) To provide an interactive, educational experience that introduces medical students the fundamental skills necessary for basic, translational, and clinical research in vision biology. The co-directors of this program request support for 6 second-year medical students each summer in vision related research laboratories that focus on topics such as visual circuitry, retinal degeneration, corneal epithelial homeostasis, uveitis, and development of the visual system. Trainees will be selected from the first year class at University of Louisville School of Medicine. Students will review research projects submitted by 19 faculty funded by the NEI or vision related foundations. The directors will make a special effort to recruit students from ethnic minorities and disadvantaged backgrounds. Students will review the available projects and enter their 1st-3rd choices, allowing matching of trainees with mentors. Final decisions will be made based on interviews between mentors and Trainees. During the 10-week summer training, students work with mentors on their research project in clinical or laboratory settings, complete training in the Responsible Conduct of Research including topics such as fabrication and falsification of data, plagiarism, managing scientific data, publication practices and responsible authorship, mentorship, stewardship, and conflict of interest. Trainees will complete human subject, IRB training, and animal care and handling as required by their specific research projects. All Trainees will attend a 2-hour weekly ?Vision Sciences Research Training Seminar? designed to introduce clinical/translational research in Ophthalmology led by mentors in this T35 program. The seminar series will culminate with each Trainee presenting his/her research project to peers and mentors. Trainees present the results of their research as posters with peers at a School of Medicine-wide, week-long celebration of research that includes nationally recognized physician- scientists as keynote speakers. Trainees will be encouraged to continue research as part of the Distinction in Research track that is an enrichment program focusing on providing meaningful and productive research experiences throughout second through fourth years of medical school, with ongoing presentations by students with mentors and 4+ weeks of research time in the fourth year toward the goal of developing clinician-researchers. Trainees will be tracked to assess the impact of this T35 program on careers as physician-scientists.

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.