Devin K. Binder - US grants

[unreadable] DESCRIPTION (provided by applicant): This project is focused on developing novel diagnostics and treatments for epilepsy based on glial cell regulation of extracellular space volume and components, and novel optical methods for seizure detection. Accumulating evidence supports a functional role for glial cells in epilepsy, at least in part via their effect on neuronal environment. Aquaporin-4 (AQP4) is the main water-selective transporting protein expressed in glial cells, and alterations in AQP4 expression in human epileptic specimens suggest that AQP4 may play a functional role in epilepsy. In recent work, I demonstrated that AQP4-deficient mice have markedly altered seizure threshold and duration. However, the regulation and function of AQP4 in the hippocampus, a structure critical to seizures and epilepsy, have not yet been studied. I propose to explore the regulation of AQP4 in the hippocampus by seizure activity and its functional role in epileptogenesis (Aims 1 and 2). Furthermore, I aim to test the hypothesis that movement of brain water can be used to develop novel optical methods for early detection of seizures (Aim 3). This proposal utilizes available mouse strains, confocal immunofluorescence and in situ hybridization, and in vivo pharmacology, electrophysiology and imaging in well-established epilepsy models. I am a fully-trained clinician-scientist specializing in epilepsy surgery and epilepsy research. I have been given the opportunity and resources for my new laboratory in the Department of Neurological Surgery at the University of California, Irvine, a world-renowned center for neuroscience and a recognized center for epilepsy research. In the training portion of this proposal, I will learn state-of-the-art optics and biophotonics techniques applied to neural tissue. My ultimate goals are to identify novel targets for antiepileptic drugs, and bridge translational and clinical research to develop optical seizure detection for use in patients. RELEVANCE: Epilepsy is a major public health problem as it is common (about 1 % of population) and causes severe neurological, psychiatric, and social disability. Current antiepileptic drugs (AEDs) are ineffective in many patients and even when effective can cause long-term cognitive impairment due to suppression of neuronal activity. Identification of glial cell-specific targets may lead to the development of novel AEDs that are effective and have fewer side effects. Glial water channels (aquaporins) are a promising target for new drug development. In addition, development of novel optical techniques for seizure detection based on changes in the brain that occur just prior to seizure onset will have a direct clinical impact on the many patients whose seizures remain uncontrolled. [unreadable] [unreadable] [unreadable]

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Epilepsy, affecting about 1% of the population, comprises a group of disorders of the brain characterized by the periodic and unpredictable occurrence of seizures. The detection and localization of seizure foci is an integral part of the treatment for epilepsy. Brainwave patterns during seizures can be detected using recording electrodes, however current technology using these recording electrodes to localize seizure activity is limited. Surface electroencephalography (EEG) can detect seizure activity with electrodes placed on the skin, but cannot provide an exact location of the seizure focus. More exact localization is possible but only through the use of an array of EEG electrodes placed directly on the brain surface across a wide area, a highly invasive surgical procedure. We propose that an alternative to electrical recording can be developed through the use of optical imaging. Our major tool of investigation is an implantable fiberoptic 850nm laser emitter paired to a fiberoptic detector that sends photons to a silicon photodiode. Our experiments have shown seizure activity produces changes in the optical scattering of the brain cortex that can be detected just before and during seizures. These optical changes correlate with seizure activity as recorded with implanted electrodes. Demonstration of this phenomenon is not only interesting from the perspective of elucidating the mechanism for the optic changes, but also as the first step in the development of minimally invasive tools for seizure detection. In this collaboration, we hope to use in vivo 2 photon imaging through a cranial window to demonstrate morphological changes (swelling) in either astrocytes labeled with GFP or sulfarhodamine 101, or mitochondria or both before and during a seizure. This will involve image acquisition and post-hoc analysis from the 2 photon laser microscope at BLI.

DESCRIPTION (provided by applicant): Numerous lines of evidence suggest that astrocytes actively participate in regulating neuronal excitability, but the role of astrocyte swelling in conrol of neuronal excitability has never been directly tested. Our long-term goal is to identify and understand astrocytic mechanisms controlling neuronal excitability. The objective in this particular application is to determine how specific manipulations of astrocyte swelling and swelling-evoked glutamate release lead to changes in neuronal excitability in situ and in vivo. The central hypothesis is that astrocyte swelling and glutamate release from astrocytic volume-regulated anion channels (VRAC) is both necessary and sufficient to elevate neuronal excitability in situ and in vivo. The rationale for the proposed research is that, identification o novel astrocytic pathways controlling neuronal excitability will provide new astrocytic drug targets for the treatment of neurological disorders and neurodegenerative disease. Guided by strong preliminary data, the central hypothesis will be tested by pursuing three specific aims: 1) Determine the extent to which astrocyte swelling-evoked glutamate release is necessary to increase neuronal excitability in situ; 2) Determine the extent to which astrocyte swelling-evoked glutamate release is sufficient to increase neuronal excitability in situ; and 3) Determine the contribution of astrocyte swelling to the control of neuronal excitability in vivo. Astrocyte swellng and glutamate release will be selectively manipulated using patch clamp and transgenic approaches, together with real-time imaging of astrocyte volume changes during recording of NMDA receptor activity in CA1 pyramidal neurons in acute hippocampal slices (Aims 1 and 2), and the effects of hypoosmolarity, hyperosmolarity and selective inhibitors on astrocytic volume changes and neuronal excitability will be assayed in vivo (Aim 3). Our approach is innovative, in our opinion, because it represents a significant departure from the status quo of assessing the role of astrocyte Ca2+-dependent gliotransmission in regulating neuronal excitability, and because techniques have been developed and proven feasible in our hands to selectively and specifically manipulate astrocyte volume changes and release of glutamate. The proposed re- search is significant, because once astrocytic mechanisms controlling neuronal excitability become clarified, novel astrocyte-directed therapies can be devised to prevent excessive levels of neuronal excitability while leaving basal levels of neuronal excitability and normal cognitive function intact. Such knowledge will also pro- vide new strategies to treat neurological disorders associated with cellular volume changes (including various forms of edema), while also fundamentally advancing our understanding of glial-neuronal interactions.

DESCRIPTION (provided by applicant): Reliable means of detecting changes which occur during the pre-seizure state could serve as a method of seizure prediction, a benchmark in epilepsy research (NIH Curing Epilepsy Conferences, 2000 and 2007). Our preliminary data indicate pre-seizure constriction in brain extracellular space (ECS) accompanied by reduction in near-infrared (NIR) optical scattering prior to detection of seizure by electroencephalography (EEG). The objective in this application is to determine the optical characteristics of the pre-seizure state in clinically relevant animal models of epilepsy. Three specific aims will be pursued: (1) To test the hypothesis that optical signals of the pre-seizure state can be used to predict epileptiform activity in vitro. Our preliminary data indicate that optical coherence tomography (OCT)-derived signals precede epileptiform activity in vitro. In this Aim, we will characterize the optical changes that occur prior to epileptiform activity in vitro in the hippocampal slice using simultaneous high-resolution microelectrode array (MEA) and OCT recordings. These experiments will fully define the optical changes occurring during the pre-seizure state and during epileptiform activity in vitro. (2) To test the hypothesis that optical signals of the pre-seizure state can be used to predict acute seizures in vivo. Our preliminary data indicate that OCT-derived reflectance intensity decreases prior to seizures in vivo (Eberle et al., 2012). In this Aim, we will test the ability of optical signal detection via OCT imaging to detect the pre-seizure state in vivo in well-established models of generalized and focal acute cortical seizures. These experiments will validate the existence of pre-seizure optical changes in distinct seizure models and provide proof-of-concept for the prediction of seizure onset in vivo with optical methods. (3) To test the hypothesis that implanted fiberoptic NIR probes can be used to detect the pre-seizure state of epileptic animals. Our preliminary data indicate that fiberoptics stereotactically implanted in mouse hippocampus demonstrate reduction in NIR reflectance prior to acute seizures in vivo. The gold standard for clinical application would be to reliably detect a spontaneous seizure in an epileptic animal. Therefore, in this Aim we will apply our novel fiberoptic NIRS detection system to a well-established animal model of chronic epilepsy (intrahippocampal kainic acid model). Sensitivity, specificity, and time course of optical NIR reflectance changes before and during chronic spontaneous seizures will be determined. These experiments will provide proof-of-principle for the efficacy of implanted fiberoptic monitoring to detect epileptic seizures for the first time. Our approach is innovative in (i) focusing on optical scattering changes rather than absorption changes as in prior studies; (ii) the first combination of MEA and OCT technologies in vitro and in vivo; (iii) use of novel fiberoptic NIR probes to measure optical changes in deep brain structures prior to seizures in vivo for the first time. The proposed research is significant because the results will elucidate optical characteristics of the pre-seizure state and lead to methods to detect focal and generalized seizures with unprecedented spatiotemporal resolution.

? DESCRIPTION (provided by applicant): The ideal neuroimaging technique would provide exquisite structural detail and also provide functional information, with high spatial and temporal resolution. Optical coherence tomography (OCT) is an optical imaging technique in which light from a low coherent source illuminates tissue and reflectivity of internal microstructures at different depths is measured by an interferometer. OCT is capable of micrometer-spatial and millisecond-temporal resolutions, without the use of exogenous contrast agents (hence label-free). The objective in this application is to develop and validate OCT for mammalian brain functional imaging; correlate OCT images with cellular electrophysiology assessed by multielectrode array (MEA); and provide proof-of- principle for OCT-based detection of neural activity. Two specific aims will be pursued: (1) Validate detection of multi-unit activity (MUA) by optical coherence tomography (OCT) in hippocampal slices. Our previous data demonstrate that OCT can detect synchronous cellular firing associated with both generalized and focal seizure activity. However, the sensitivity of OCT to physiological events such as multiunit activity (MUA) has not yet been determined. In this Aim, MUA induced by 4-aminopyridine (4-AP) and high K+ will be correlated to OCT. (2) Validate detection of local stimulation-induced synaptic activation by OCT. To allow more precise control of local stimulation site and intensity, in this Aim we will use local stimulation of a defined synaptic pathway in the hippocampal slice combined with OCT-based detection. Stimulation of the Schaffer collateral pathway from CA3 to CA1 will be performed by (1) electrical stimulation and (2) optogenetic stimulation to trigger MUA in CA1 that will be then detected by MEA and correlated to the changes in the optical signal by OCT. Our approach is innovative in adapting OCT for brain functional imaging. The proposed research is significant because it will lead to the validation of OCT as a neuroimaging tool for research in neuroscience.

Cognitive impairment occurs and is more prevalent during primary progressive MS[1]. While MS clinical presentation is protean, epidemiological studies have revealed that MS patients are three to six times more likely to develop epileptic seizures than the population at large. Excitotoxic neuronal damage in the hippocampus (and other regions) is thought to be one of the causes for cognitive deficits in nearly 50% of multiple sclerosis (MS) patients and could be due glutamate dyshomeostasis. Glutamate is a major excitatory neurotransmitter in the mammalian CNS. Our recent published results have shown i) a decrease in inhibitory parvalbumin neurons of chronic cuprizone-diet fed demyelinating mice (Lapato et al., 2016) and in the hippocampus of MS patients with seizures (Lapato et al., 2020); ii) astrocyte glutamate uptake and water homeostasis are dysregulated in the hippocampus of MS patients with seizures (Lapato et al., 2020)[4]. The objective of this application is to understand how demyelination-induced loss of inhibitory neurons impacts hippocampal changes that lead to learning and memory deficits. We hypothesize that chronic demyelination induces decrease in hippocampus PV neurons and indices substantial changes in synaptic transmission involved in learning and memory. In aim 1: we will determine chronic cuprizone diet-demyelination induced changes in long term potentiation by electrophysiology in brain slices. In aim 2, we will examine synaptic changes during chronic demyelination in the CA1 and striatum radiatum regions of the hippocampus. In aim 3, we will assess chronic demyelination induced changes in learning and memory. We anticipate that this research will be transformative, as we will introduce to the research community a functional and molecular mechanism for memory disorder due to demyelination.