John R. Huguenard, Ph.D. - US grants

The corpus callosum provides a pathway for contralateral projections that leads to generalization of epileptic activity. In humans and in animal models of epilepsy, callosotomy has been successfully employed to decrease the severity and frequency of seizure activity. Virtually all of the callosal projections are excitatory onto excitatory cells. Control of synaptic strength in these projections is likely to be an important mechanism in determining the strength and generalization of seizure-related electrical activity in the cerebral cortex. For example, if callosal synapses fatigue they may lose their effectiveness in transmitting epileptiform activity. Furthermore, neuromodulator-mediated actions at synaptic terminals will likely decrease release of excitatory neurotransmiters and may reduce the spread or initiation of epileptiform activity. Little is known of the specific release mechanisms in the callosal pathway. In this proposal, the development of specific callosal connections and their neuromodulation will be examined. We will use in vitro slices of frontal and parietal cortex from rat that contain callosal fibers within the section of the slice. Whole-cell patch clamp methods will be employed to record isolated excitatory synaptic currents from visually identified infragranular pyramidal neurons. Presynaptic neuromodulatory effects of acetylcholine and norepinephrine on callosal synapses will be examined and compared to a control synapse: excitatory connections arising outside the cortex in the thalamus. The results of this study will lead to information regarding the potential role of cortico-cortical connections in the generalization of cortical electrical activity during seizures.

DESCRIPTION The specific neuronal circuitry that provides the foundation for sleep-related electrical activities in the brain is composed of reciprocal loops between interconnected areas of thalamus and neocortex. There is emerging evidence that this circuitry can be compromised in humans with generalized absence epilepsy, with the result that normal oscillatory sleep rhythms are converted into pathological epileptic activities. Within the thalamic half of the thalamocortical loop synaptic mechanisms promote both normal and abnormal oscillatory patterns. It is our hypothesis that regulation of synaptic strength within the thalamus is a key factor in determining whether normal are transformed into those associated with absence epilepsy. As a first step in testing this hypothesis, presynaptic control mechanisms in the thalamus will be investigated. These presynaptic mechanisms are expected to reduce the efficacy of synaptic transmission and may play a key role in the prevention of the neuronal activity associated with absence seizures. Gamma-aminobutyric acid (GABA receptor agonists and antagonists are powerful regulators of experimental absence seizures. The presynaptic actions of these compounds will be examined in the two principle types of neurons in rat somatosensory thalamus, the inhibitory cells within nucleus Reticularis thalami (nRt) and the excitatory relay neurons in the ventrobasal complex (VB). A microscope fitted with differential interference optics will be used to obtain high quality whole-cell voltage-clamp recordings form neurons visualized in thalamic slices maintained in vitro. The results of these studies may lead to new approaches in the treatment of human generalized epilepsy.

DESCRIPTION: (Verbatim from the Applicant's Abstract) It is becoming increasingly clear that similar neuronal networks are activated both normally during sleep and pathologically during the thalamocortical seizures that occur in generalized absence epilepsy. What determines whether the thalamocortical system acts in a normal or a pathological manner? Recent studies pinpoint the thalamic reticular nucleus as a key regulatory site in this process. The reticular nucleus is poised to intercept and act upon the ascending and descending flow of cortical information. The reticular nucleus is poised to intercept and act upon the ascending and descending flow of cortical information. Output from reticular neurons serves to inhibit thalamocortical relay neurons and shape sensory responses as well as to contribute to the generation of thalamocortical oscillations related to sleep and epilepsy. In mice with an inhibitory neurotransmitter receptor gene (GBRB3) inactivated, neurons in the thalamic reticular nucleus lose their ability to inhibit each other. This loss of inhibition is associated with a dramatic increase in epileptiform synchrony measured in vivo and in vitro with thalamic brain slices. Intra-reticular inhibition thus seems to normally produce distance-dependent differences in the timing of electrical responses across the extent of the thalamus. Elimination of such timing differences leads to pathological synchrony. In addition to structural changes in intra-reticular inhibition, dynamic changes, such as down-regulation via activation of presynaptic GABA B receptors can occur. This would promote seizures, but other GABA B receptor actions have the opposite effect. In this proposal, molecular, computational , and neurophysiological approaches, including knockdown and knockout experiments, will be used to test the hypothesis that recurrent connectivity between thalamic reticular cells prevents the hypersynchrony of absence epilepsy, and that collapse of the interconnectivity can promote seizure generation . To address the latter question, we will develop a strategy to specifically antagonize GABA B receptors that promote seizures, with the ultimate goal of improved treatment of absence epilepsies.

DESCRIPTION (provided by applicant): The AMPA class of glutamate receptors mediate the majority of excitatory neurotransmission in the brain. These receptors are specifically implicated in the propagation of seizure activity. One member of the AMPA receptor family, the GluR2 subunit, dominates connections between excitatory neurons in the mature neocortex. The absence of this subunit alters the properties of AMPA receptors such that they become impermeable to calcium ions and show use-dependent facilitation. Down-regulations in GluR2 have been noted in a number of animal epilepsy models and in human epilepsy. This laboratory has recently demonstrated that GluR2 is functionally expressed in pyramidal neuron synaptic receptors at very low levels early in rat neocortical development. Thus it appears that in epileptic cortex there may be a recapitulation of the early postnatal phenotype of GluR2-lacking receptors, which are hypothesized to be epileptogenic in adult brain. In this proposal, whole cell voltage-clamp techniques and laser-scanning caged-glutamate photolysis will be used with rat neocortical brain slices to test the generality of the finding regarding developmental GluR2 alterations in pyramidal neurons of different cortical regions and lamina. Further, the functional consequence regarding the ability of excitatory synaptic circuits to sustain repetitive, seizure-like activity will be examined. The hypothesis will be tested that decreased expression of GluR2 subunits in epileptic tissue results in a functional alteration in the synaptic receptors that would allow for increased calcium entry and post-synaptic facilitation, both of which may be important in the development of epilepsy.

[unreadable] Description (provided by applicant): Penetrating Injuries of the brain are a frequent cause of epilepsy in man, making it important to understand the underlying pathogenetic mechanisms. Loss of inhibition has been found in a number of models of epilepsy and may be important in posttraumatic human seizure disorders. The specific aims of the proposed experiments focus on two types of abnormality, found in a group of inhibitory cells within the partial cortical isolation model of posttraumatic epilepsy. These fast-spiking (FS) interneurons have a major influence on the control of runaway activity in the cortex which, if unchecked, can lead to epileptic seizures. Anatomical changes in the axons of FS cells suggest that they make fewer functional contacts that would release GABA on themselves via "autaptic1 synapses, and on excitatory pyramidal cells. They also have reductions in a vital enzyme, the sodium pump. In vitro slices from chronically injured epileptogenic cortex together with patch clamp techniques and dual recordings will be used to assess the functional disorders in inhibition that occur as a result of these axonal abnormalities. The long term goal of such experiments is to uncover links between injury and the appearance of epilepsy that can be modified by strategies for prevention or treatment, such as development of targeted drugs. For example, if reductions in the "sodium pump" contribute to cortical hyperexcitability and epileptogenesis, if may be possible to use pharmacological agents or neurotransmitters to boost pump activity and ameliorate seizure activity. The discovery that important cortical inhibitory neurons are "disconnected" from their targets after injury, rather than being killed, may open the way for approaches that will promote "rewiring" of these connections to restore the balance between excitatory and inhibitory processes in the damaged areas. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The thalamus is a subcortical structure that is widely interconnected with cortical circuits and plays a critical role in such vital and diverse brain functions as processing of sensory information, sleep and memory. Disruptions in thalamic structure and function are implicated in both generalized and focal epilepsies, yet certain aspects of thalamic function may serve to constrain epileptiform activity. Our preliminary results suggest mechanisms through which thalamic circuits provide activity-dependent adaptive and maladaptive changes that suppress or enhance epileptogenesis, respectively. In particular, cortical infarction or generation of focal cortical epileptiform activity leads to maladaptive increases in thalamic network connectivity and function that would enhance epileptogenesis. By contrast, multiple lines of evidence suggest that endogenous ligands for the benzodiazepine site of inhibitory GABA receptors, endozepines, are constitutively expressed in the thalamus to augment synaptic inhibition specifically in the thalamic reticular nucleus, a site proposed to underlie a key seizure regulatory pathway. In addition, the endozepine effects appear to be increased by experimental absence seizures, and act to blunt seizure intensity and duration. The proposed experiments will employ anatomical and electrophysiological approaches to analyze each mechanism individually in addition to their interactions, with the long term goal of providing an integrated view of adaptive and maladaptive processes important in the roles of this key brain structure in normal brain function and epilepsy.

DESCRIPTION (provided by applicant): Synaptic interactions within the thalamus are dynamically regulated in terms of their strength and efficacy. When this dynamic regulation fails to keep synaptic strength in the normal operating range, the thalamocortical circuitry enters a hyper synchronous state that leads to the development of generalized absence seizures. Inhibitory synapses mediated by the neurotransmitter gamma-aminobutyric acid (GABA) play a critical role in regulating synchrony, especially those synapses mediating reciprocal inhibition between thalamic reticular neurons. The long term goals of this project are to understand the pathways that lead to failure of the reciprocal inhibitory circuit and to design interventions that will prevent seizures. In this proposal we will address three major questions relevant to the efficacy of these inhibitory synapses: 1) What are the roles of glial GABA uptake via the transporter GAT-3 and glutamine-dependent GABA cycling in the context of epileptic network oscillations? 2) What is the extent and potency of specific inhibitory connections within the thalamic reticular nucleus? and 3) Can the reciprocal inhibition between thalamic reticular cells be functionally overcome by excitatory corticothalamic and/or thalamocortical synaptic responses? Overall these three questions will address the central theme - synaptic and perisynaptic factors that critically regulate excitability in the reticular nucleus. Our approach will be a combination of whole-cell voltage and current clamp recordings of thalamic neurons in acute rat brain slices, combined with laser scanning photolytic glutamate uncaging, dynamic clamp, genetically-modified mice, and pharmacological manipulation of the GABA neuron-glial transport system. The results of these experiments will provide insight regarding therapeutic approaches that are likely to be effective in the treatment of generalized absence seizures.

DESCRIPTION (provided by applicant): The aim of the Neurosciences Graduate Program at Stanford University is to develop predoctoral PhD students as leaders in neuroscience research and teaching. We propose an integrated educational program that involves each student in the study of all levels of nervous system function from molecules to behavior. Teaching students how to identify, approach and solve specific research problems will promote their professional development as independent scientists and will contribute new knowledge to the fight against neurological and psychiatric disease. To this end the Program will provide students with the opportunity to conduct state-of-the-art neurobiological research in any of a broad range of disciplines including molecular and cell biology, genetics, biophysics, electrophysiology, anatomy, computational modeling, neuroimaging of the human brain, and the quantitative study of behavior. Formal course work will require students to examine how the nervous system functions at the molecular and cellular level, during development from embryo to adult, and in normal and diseased states. The Program incorporates added depth and breadth via a suite of activities including retreats, seminar series, summer courses, and invited lecturers. There are currently 46 students in the Program. All students will be enrolled in the Interdepartmental Program in Neuroscience, the only academic body at Stanford that awards a PhD in the neurosciences. The faculty is composed of 67 members from 14 Departments in 3 Schools. The faculty is highly interactive, intellectually diverse, and their research efforts are well funded. Their research covers nearly every aspect of neuroscience. Trainees are encouraged to rotate through three labs before committing to a preceptor. Course requirements must be fulfilled with courses taught by different academic departments, and the members of the examination and thesis committees must be from more than one department. The Program Committee, which is the governing body, is composed of Program faculty from 7 departments. This Committee has overall responsibility for setting academic policy, selecting students and monitoring their progress after matriculation. Admitted students are among the most outstanding candidates in the nation. Past trainees of the Neurosciences Program have been extremely successful in pursuing academic research careers.

DESCRIPTION (provided by applicant): This application requests renewed support for an institutional postdoctoral training program in neuroscience and epilepsy that will allow faculty in the Departments of Biological Sciences, Molecular and Cellular Physiology, Comparative Medicine, Neurology and Neurological Sciences, Neurobiology, and Psychiatry at Stanford University to attract fellows to careers in research areas ultimately relevant to the problems of epilepsy in man. The faculty employ the methods of cellular neurophysiology and biophysics, biochemistry, molecular biology, neuroanatomy, neuropharmacology, and the use of animal model systems for studies of normal and abnormal structure/function. Faculty research interests include cortical neuronal and glial development;physiological and morphological changes in nerve cells and circuits in animal models of chronic neocortical and hippocampal epileptogenesis;effects of drugs and transmitters on neurons and network activity in cortex and thalamus;development, organization, and synaptic physiology of the CNS, especially neocortex;cellular and molecular aspects of long-term changes in neuronal excitability;the role of gene structure, expression and modulation on neuronal function;and the effects of trophic factors in the normal and diseased nervous system. Trainees may learn techniques of neurophysiology and neuropharmacology applied to neurons in slices or cultures, including patch-clamp methods for biophysical studies and assessment of transmitter actions;use of in vivo and in vitro animal models of epileptogenesis;anatomic techniques for intracellular labeling, immunocytochemistry and in situ hybridization;cell culture;molecular techniques including gene isolation and cloning, site directed mutagenesis;clonal analysis;cell transplantation;experimental gene therapy;and use of transgenic animals. A variety of available courses and seminars contribute to the breadth of research training. Participation of clinical department faculty fosters effective research interactions between MD and PhD trainees and a focus on the interface between basic neuroscience and clinical issues requiring investigation. The positions are advertised nationally and applicants solicited in accord with, and in the spirit of affirmative action.

Epilepsy results from abnormal recruitment of recurrent excitatory synaptic circuits in the neocortex. The long term goals of this project are to determine the mechanisms underlying epileptic recruitment and to develop therapies that will prevent it. In the developing brain excitatory neocortical networks are synaptically connected via an immature form of postsynaptic ionotropic glutamate receptor that lack the GluR2 receptor subunit. Synapses lacking this subunit show dynamic forms of synaptic plasticity not normally seen in mature neurons, which prominently express synaptic GluR2 receptors. Our preliminary data suggests that in the cortical freeze lesion (microgyrus) model of focal cortical dysplasia, neocortical pyramidal neurons express immature forms of synaptic glutamate receptors and display prominent epileptiform hyperexcitability that is recruited by focal molecular uncaging of glutamate. In the first aim of the proposed experiments we will use in vitro electrophysiological means to determine the mechanisms underlying BDNF- and PKC- mediated plasticity of synaptic receptors in immature neocortical circuits. In addition we will test whether such plasticity exists in synapses of the microgyrus model. In the second aim we will utilize laser scanning photostimulation to determine the mechanisms underlying the abnormal recruitment of epileptiform electrical responses in the microgyrus model. The results of these studies will provide information relevant to our understanding of the mechanisms underlying focal cortical seizures, and may indicate therapeutic targets for antiepileptic drug development.

The core will provide critical administrative and technical support for all projects. Administrative tasks include organizing lab meetings and retreats, maintaining lab records, complying with various regulatory tasks, ordering and maintaining supplies, etc. The core also provides excellent molecular biological, neuroanatomical and shop services that support all projects. The core maintains a completely equipped histology lab and mechanical and electronics shop.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Many factors ranging from neurotransmitter release to receptor localization to neurotransmitter uptake mechanisms will affect GABA-mediated synaptic inhibition, especially for metabotropic (GABAB-mediated) responses. As postsynaptic GABAB receptors are localized primarily outside of synaptic sites (ie extrasynaptic), it is hypothesized that GABA transporters (GATs), which function to remove extracellular GABA, limit the extent of GABA spillover and, therefore, GABAB receptor activation. To better understand how GATs regulate GABA spillover, we are examining the roles of two GATs, GAT1 and GAT3, in defining features of GABAB IPSCs in the rat thalamus. Our electrophysiology data demonstrate that GAT1 and GAT3 differentially regulate the amplitude and kinetics of GABAB IPSCs. Our anatomical data suggest that these different GAT1/3 actions result from differences in subcellular localization and density of the two GATs. Specifically, GAT1 expression is primarily perisynaptic, while GAT3 is found in both peri- and more distal extra-synaptic regions. We are beginning to explore how such differential localization can influence GABAB currents. We are currently using computational approaches to explore how GAT localization regulates GABA diffusion in the thalamus, and how such regulation determines the properties of GABAB IPSCs. Specifically, we are using MCell, a modeling platform that tracks the stochastic nature of diffusing molecules in 3-dimensional microphysiological environments using Monte Carlo algorithms (Stiles and Bartol, 2001). Our simpler, less computationally-intensive models indicate that differential GAT1/3 localization provides a mechanism by which GABA transients can be modulated to enable distinct GABAB IPSC amplitude and kinetic changes. We now would like to verify these initial findings in more complex models. Dr. Joel Stiles suggested that we utilize resources offered by the Pittsburg Supercomputing Center to run our new models. Therefore, in following his suggestion, we are applying for these resources to complete our project. We would like user accounts for Mark Beenhakker and John Huguenard

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. Salk Friendly grant

DESCRIPTION (provided by applicant): The aim of the Neurosciences Graduate Program at Stanford University is to develop predoctoral PhD students as leaders in neuroscience research and teaching. We propose an integrated educational program that involves each student in the study of all levels of nervous system function from molecules to behavior. Teaching students how to identify approach and solve specific research problems will promote their professional development as independent scientists and will contribute new knowledge to the fight against neurological and psychiatric disease. To this end the Program will provide students with the opportunity to conduct state-of-the-art neurobiological research in any of a broad range of disciplines including molecular and cell biology, genetics, biophysics, electrophysiology, anatomy, computational modeling, neuroimaging, and the quantitative study of behavior. Formal course work will require students to examine how the nervous system functions at the molecular and cellular level, during development from embryo to adult, and in normal and diseased states. The Program incorporates added depth and breadth via a suite of activities including a laboratory boot camp, retreats, seminar series, summer courses, and invited lecturers. There are currently 80 students in the Program. All students will be enrolled in the Interdepartmental Program in Neuroscience, the only academic body at Stanford that awards a PhD in the neurosciences. The faculty is composed of 85 members from 19 Departments in 3 Schools. The faculty is highly interactive, intellectually diverse, and their research efforts are well funded. Their research covers nearly every aspect of neuroscience, with concentrations in cellular/molecular, computational, developmental, systems/cognitive/behavioral neuroscience, membrane excitability and neurobiology of disease. Trainees are encouraged to rotate through three labs before committing to a preceptor. Course requirements must be fulfilled with courses taught by different academic departments, and the members of the examination and thesis committees must be from more than one department. The Program Committee, which is the governing body, is composed of Program faculty from eight departments, along with student representatives. This Committee has overall responsibility for setting academic policy, initiating programmatic changes, and monitoring student progress after matriculation. Admissions and curriculum issues are handled by separate committees, each composed of similarly diverse faculty/student groups. Admitted students are among the most outstanding candidates in the nation. Past trainees of the Neurosciences Program have been extremely successful in pursuing academic research careers.

Project Summary: Presynaptic mechanisms controlling excitatory and inhibitory synaptic transmission in the cerebral cortex have critical roles in normal information processing and also may contribute to the pathophysiology of a variety of brain disorders such as cognitive decline and epilepsy. The specific aims of these experiments focus primarily on inhibitory synaptic transmission mediated by gamma-amino butyric acid (GABA)-containing inhibitory interneurons and its regulation by 3 potent and ubiquitous processes in normal cerebral cortex and in a model of posttraumatic epileptogenesis. These neurons are known to be vulnerable to injury. Specific aims relate to (1) control of transmitter release by presynaptic Ca++ channels and (2,3) modulatory effects on GABAergic inhibition produced by actions of neuropeptide Y and GABA at their receptors and selective Ca++ current blockers on presynaptic terminals of major classes of inhibitory interneurons. Techniques employed include use of whole cell patch clamp recordings of spontaneous and evoked inhibitory postsynaptic currents (IPSCs) generated by identified subclasses of interneurons in in vitro brain slices; laser scanning photostimulation to map cortical connectivity; paired recordings to examine unitary IPSCs from interneurons to other interneurons and pyramidal cells; use of genetically engineered mice with GFP label in specific interneuron species; and local application or bath perfusion of receptor agonists and antagonists. The partial cortical isolation model will be used to provide chronically injured, epileptogenic neocortical slices and assess changes in these presynaptic modulatory mechanisms that might contribute to hyperexcitability. The long term goals are to identify critical abnormalities that might eventually be targets for selective agents that would used to prevent or treat human posttraumatic epilepsy.

DESCRIPTION (provided by applicant): We are requesting NINDS support for a Gordon Research Conference on Mechanisms of Epilepsy and Neuronal Synchronization to be held August 8-13, 2010 at Colby College in Maine. The main goal of the study of seizures is to identify the mechanisms underlying synchronous electrical discharges in neuronal networks in order to develop more effective and less toxic treatments and cures for epilepsy. A unique, intellectually challenging aspect of epilepsy research arises from the fact that it encompasses virtually all major levels of biological organization, from genes and ion channels to circuits and behavior. The major purpose of this Gordon conference is to bring together geneticists, molecular biologists, developmental neuroscientists, neuroanatomists, electrophysiologists, and computational neuroscientists working on basic mechanisms related either directly or indirectly to seizure generation to synthesize current advances and set the stage for future discoveries. The theme of the current conference is Neural Circuits and Epilepsy, and topics to be covered include: Microcircuit Dynamics: Emergence of synchrony;Inhibitory neurons: failure to deliver;Neuroinflammation and Epilepsy;Circuit Homeostasis: Analytical and Experimental approaches in Epilepsy;Epigenetics, gene regulation and microRNAs in Epilepsy;Circuit development and function: basis for epileptic networks;Stem Cells and Epilepsy;Epileptogenesis: occult and overt mechanisms;and Epileptic ion channels and synaptic proteins. Our goals are to disseminate the latest scientific advances, foster productive new insights and collaborations, and set the stage for new translational studies that will bring the newest discoveries to the bedside in the shortest possible time. PUBLIC HEALTH RELEVANCE: Epilepsy is a chronic condition that affects 50 million people worldwide. Development of novel therapeutic strategies to better treat and potentially cure this devastating condition requires scientific advances to better understand the molecular and cellular mechanisms responsible for epilepsy. This conference will bring together experts from all over the world in an intensive, immersive environment to present and discuss novel findings, facilitating dissemination of knowledge and spawning collaborations. This should significantly advance research directed towards better treatment of epilepsy.

? DESCRIPTION (provided by applicant): This application requests renewed support for an institutional postdoctoral training program in epilepsy research. Epilepsy is a complex disease requiring an integrated multidisciplinary approach designed to effectively train future research leaders in the field. Accordingly faculty with a wide range of relevant expertise in the Departments of Biological Sciences, Molecular and Cellular Physiology, Comparative Medicine, Neurology and Neurological Sciences, Neurobiology, Neurosurgery, and Psychiatry at Stanford University have been assembled to create a training program that attracts fellows to careers in research areas especially relevant to the problems of epilepsy in man. The faculty employ modern neuroscience approaches including live imaging, cellular neurophysiology, optogenetics, biochemistry, genetics, neuroanatomical approaches, and the use of animal model systems for studies of normal and abnormal structure/function. Faculty research interests include cortical neuronal and glial development and function; physiological and morphological changes in nerve cells and circuits in animal models of chronic neocortical and hippocampal epileptogenesis; dissection and intervention of neuronal microcircuits implicated in seizures and epileptogenesis; development, organization, and synaptic physiology of the CNS, especially neocortex, thalamus, hippocampus; cellular and molecular aspects of long-term changes in neuronal excitability; and the roles of gene structure, expression and modulation on neuronal function, especially interneurons. Trainees may learn techniques of whole animal EEG, behavior, and intracranial recording; optogenetics; neurophysiology in reduced preparations such as slices or cultures; anatomic techniques for intracellular labeling and tract tracing, immunocytochemistry and in situ hybridization; cell culture; cell transplantation; experimental gene therapy; and use of transgenic animals. The training program consists of monthly integrative sessions, including seminars, didactic lectures, and clinical content, all focused on epilepsy. Participation of clinical department faculty fosters effective research interactions between trainees and a focus on the interface between basic neuroscience and clinical issues requiring investigation. The positions are advertised nationally and applicants solicited in accord with, and in the spirit of recruiting individuals from diverse backgrounds.

DESCRIPTION (provided by applicant): We have recently shown that naturally occurring neuroactive compounds, endozepines related to the protein Diazepam Binding Inhibitor (DBI) in the brain can mimic the activity of benzodiazepines, which are effective treatments in epilepsy. Benzodiazepines are allosteric modulators of GABAA receptors, which mediate the primary form of synaptic inhibition in the brain. This suggests that, through production of endozepines, brain cells and circuits can-self regulate, to dynamically enhance synaptic inhibition as needed to suppress seizures as they arise from ongoing brain activity. While the existence of DBI-related antiepileptic endozepines has now been demonstrated, little is known regarding the cellular source of endozepines, nor of the means through which they are secreted from cells or processed in the extracellular space to exert their action. The proposed experiments have three aims. 1) Determine whether astrocytes are the source of endozepines, as they express very high levels of DBI, and appear to have a high capacity for secretion, 2) determine the pathways through which cells, most likely astrocytes, sense activity and then respond through secretion and processing of DBI, and 3) Identify the final endozepine molecule (or molecules), which does not appear to be DBI itself, but a protein fragment of DBI. We will use electrophysiological assays to document functional endozepine activity in all three aims. Assays will include determining the kinetics of spontaneous inhibitory post-synaptic currents, an effective assay for detection of exogenous or endogenous allosteric modulation of synaptic GABAA receptors, and responses to high-speed iontophoretic GABA application, a sensitive assay for endozepine activity, network analysis of large-scale thalamic networks in vitro, and EEG analysis of seizure susceptibility in vivo. These studies will be facilitated by the availability of mutant mice that alow for targeted deletion of DBI from astrocytes, and by fluorescent reporter mice that allow for detection of the range and extent of deletion. Overall the results of the proposed experiments will provide mechanistic information regarding endozepine signaling and whether this natural brain activity might ultimately be targeted for therapeutic intervention in epilepsy and other neuropsychiatric disorders of altered GABA signaling.

DESCRIPTION (provided by applicant): Autism spectrum disorders (ASDs) have risen to approximately 1 in 88 in the Unites States over the past years, affecting an entire generation of children, families and communities. Currently, the diagnosis for most forms of ASD is based on a triad of behavioral symptoms, including social impairments, communication difficulties, and repetitive or stereotyped behaviors, with no quantitative measures for screening or assessment of potential drug therapies. Electrophysiological measurements of synapses and neuronal networks from these patients may hold the potential for diagnosing, characterizing and analyzing the effectiveness of potential treatment strategies. Here, we propose to apply a transformative technology for the long-term intracellular recording networks of neurons differentiated from patient-derived iPSC. To accomplish this goal, we have created a solid-state device comprised of 2D arrays of Stealth electrodes that sit passively within the membrane of neuronal cells and have the capacity to record synaptic, neuronal and network properties of multiple interconnected neurons simultaneously for days to weeks. Through the optimization of the fabrication of these Stealth probes and the transformation into a turn-key device, we will evaluate the feasibility of this platform as a diagnostic and research tool for ASD. We then propose to use this innovative scalable analytical platform to characterize the neuronal, synaptic and network signatures of neurons differentiated from iPS cells derived from patients with Phelan-McDermid Syndrome and then assess the effectiveness of emerging drug therapies to normalize aberrant signatures. If successful, our solid-state platform can be transformed into a high-throughput screening device that will allow investigators to recapitulate early developmental stages of ASD and evaluate the effects of ASD mutations and environmental insults on neuronal network and synaptic function and utilize this as a tool for drug screening, diagnosis and personalized treatment.

The brain has likely evolved multiple control mechanisms to regulate activity and keep it in a robust operational state. Occasionally these regulatory mechanisms break down, and uncontrolled activity in the form of epileptic seizures ensues. This is especially evident in the syndrome of primary generalized epilepsy in which global seizures suddenly arise from a normal behavioral state. A form of primary generalized epilepsy, absence epilepsy, is expressed in a network composed of widespread regions of neocortex and a subcortical structure, the thalamus, that together form the thalamocortical circuit. Childhood absence seizures are characterized by widespread synchronized thalamocortical activity, EEG 3/s spike and wave discharge, and loss of consciousness. Validated genetic rat and mouse models of absence epilepsy have identified some of the thalamic and cortical microcircuit elements, i.e. the individual neuron types and their synaptic connections that participate in the epileptic network, yet it remains unclear how the circuit suddenly and unpredictably switches its state from that of normal functioning to seizure generating and back. Recent evidence suggests that in one central node of the thalamocortical network, the reticular thalamus (RT), a single type of branch of RT neuron axonal output is specifically susceptible to sporadic failures that might explain sudden seizure onset. Pilot data indicate that this internal branch, which regulates RT itself, can fail in a use-dependent way that would lead to uncontrolled RT activity that can precipitate seizures. Further, Scn8a deficient mice, with frequent absence seizures, show increased intra-RT failures, indicating a causative role. Only recently have the methods become available to directly study axon function, allowing us for the first time ask questions about how the selective failure of neurotransmission in axon branches can occur. Experiments will utilize high resolution 2 photon imaging and electrophysiology to visualize the different branches of RT axons and address the novel hypothesis that failure, i.e. the inability to send efferent synaptic signals, through individual output axon branches and their synaptic release sites could be causative in epilepsy. Aims will determine the conditions in which selective branch failure of intra-RT vs RT output axons to the dorsal thalamus occurs, and the mechanisms for the failure, whether they be through failure of action potential generation or through decreased probability of synaptic release, and whether failure mechanisms may apply more broadly to epilepsies. The results of these studies could inform the development of potential new epilepsy treatment approaches that would prevent the failure of key output branches.

PROJECT SUMMARY/ABSTRACT In the general population, less than 1% of children develop seizures, whereas over 30% of children with autism spectrum disorder (ASD) do so by adolescence. Maternal infection during pregnancy is a risk factor for both ASD and epilepsy, however we have limited understanding of how early-life immune insults alter neural circuitry implicated in both disorders. The medial prefrontal cortex (mPFC) is a brain region important for regulating diverse cognitive and emotional behaviors altered in ASD. We have obtained pilot data supporting altered mPFC circuit function after gestational exposure to a viral mimetic polyinosinic:polycytidylic acid (poly(I:C)). Using a high-throughput multisite field potential recording approach in acute mPFC brain slices, our preliminary results indicate that maternal immune activation (MIA) leads to persistent alterations in mPFC activity, at both presynaptic and postsynaptic loci, and an increased susceptibility to generation of epileptic activities in vitro. The proposed experiments will elucidate the precise cell type- and lamina-specific alterations underlying changes in network excitability. Aim 1 of this proposal will identify changes to functional intracortical connectivity within the mPFC. Pregnant dams will be treated with saline or poly(I:C) to mount an acute antiviral inflammatory response. Acute brain slices containing mPFC will be prepared from adult offspring at three to four months of age. Laser scanning photostimulation based on patterned glutamate uncaging will be used to map excitatory synaptic connectivity within the mPFC between photo-excited presynaptic neurons and individual postsynaptic pyramidal and interneurons. Changes to laminar-specific inputs to excitatory and inhibitory cells types following MIA will be assessed by intracellular voltage-clamp recordings of synaptic activity. Immunohistochemical methods will confirm cell type, location and morphology and will be used to identify structural defects in lamination. Aim 2 will evaluate alterations in the recruitment of distal inputs to the mPFC from two regions highly implicated in the initiation and spread of limbic seizures, the basolateral amygdala (BLA) and ventral hippocampus (vHC). AAV-CamKII?-ChR2/eYFP will be used to target expression of channelrhodopsin-eYFP or eYFP alone to excitatory neurons in these extra-prefrontal structures. Postsynaptic currents in mPFC in L2/3 and L5 neurons will be measured in response to optogenetic activation of BLA and vHC terminals, respectively, and changes in excitatory and inhibitory components assessed. Together these aims address circuit intrinsic and extrinsic changes that may contribute to the shared pathogenesis of ASD and epilepsy. Information gained from the interrogation of mPFC circuitry in an established MIA model will identify neuropathological alterations that might be targeted for therapies.

Voltage Gated Sodium Channels (VGSCs) are the fundamental molecular basis of neuronal excitability. VGSCs render neurons as exquisite sensors of incoming synaptic signals that can be amplified to generate what are classically thought of as ?all-or-none? output signals - action potentials (APs). VGSC functions include initiation and propagation of APs, promotion of repetitive or burst spikes, and boosting of synaptic currents,- actions that depend on localization to distinct subcompartments of the neuron. However, manipulating VGSC function and therefore membrane excitability of spatially confined subcompartments of cells is challenging given currently available tools. In response to this need, we have developed a class of synthetic photocaged saxitoxin compounds (STX-PC) that enable fine spatial, temporal, and reversible control of membrane excitability via VGSC block. We propose 1) to develop first and second generation STX-PC variants for single- and multi-photon uncaging and to characterize their performance for in vitro and in vivo applications, 2) to provide proof of concept evidence that these tools are validated to assess defects in macro- and micro-scale VGSC-mediated axonal AP propagation in wild-type versus genetic epilepsy models with altered VGSC (Scn8a) function. At the macro scale we will test the ability of uncaged STX-PC to block AP propagation in the major axon fiber tract of the corpus callosum in vitro and in vivo. At the micro scale will validate the use of STX-PC to study the roles of VGSCs in defined subcompartments of the cell (e.g., axon initial segment) in promoting spike initiation and back propagation. These tools also have therapeutic potential, which will be piloted in Scn8a mice. This work we view as the critical step in creating and validating tools that can be subsequently used by researchers asking mechanistic questions about VGSC function in normal physiological activity and how this is disrupted in neuropsychiatric diseases. A prime example would be epilepsy in which mutant VGSCs with both gain- and loss-of-function are associated with a variety of seizure disorders.

Maternal infection during pregnancy is an established risk factor for Autism Spectrum Disorders (ASD), yet we have limited knowledge of how immune insults alter neural circuitry in the developing brain and lead to profound behavioral impairment. The medial prefrontal cortex (mPFC) plays a critical role in the higher-order sociocognitive functions compromised in ASD. We obtained an exploratory R21 grant to develop a large-scale, silicon probe-based analysis of mPFC circuitry in adult offspring following maternal midgestational exposure to a viral mimetic polyinosinic:polycytidylic acid (poly(I:C)). Using this high-throughput approach, we identified an unexpected hypofunction in the output fibers of layer 5 projection neurons as the central defect in mPFC. In particular, layer 5 axons were less able to sustain output during prolonged activity, and temporal precision was impaired. Transcriptomic profiling of the mPFC revealed a marked deficiency in the cell adhesion molecule L1cam, a putative regulator of axonal excitability. The proposed work will explore the functional ramifications of axonal hypoactivity on specific long-range targets of the mPFC ? mediodorsal thalamus (MD), basolateral amygdala (BLA), and nucleus accumbens (NAc) ? that also mediate behaviors dysregulated in ASD. Aim 1 of this proposal will test whether direct recruitment and/or indirect feedforward inhibition of principal cells in these subcortical regions is impaired in offspring exposed to maternal immune activation (MIA). Layer 5 projections will be specifically targeted for optogenetic stimulation with viral approaches. In Aim 2, we will augment layer 5 output with complementary genetic (L1cam rescue) or optogenetic (SSFO-driven enhanced excitability) approaches to determine whether this restores L5 output and rescues ASD-related deficits. In Aim 3, we will test whether somatosensory cortex, another cortical region implicated in ASD behaviors, is similarly affected, as would be expected if midgestational MIA exposure broadly affects layer 5 cortical neurons at a critical step in their genesis and maturation. Experiments will test for axonal hypofunction, and altered output to a site of sensorimotor integration, the posterior medial (POm) thalamic nucleus. The results of the proposed experiments have implications regarding improved understanding of ASD pathogenesis, as well as identifying a molecular pathway that could be targeted for restored function. !

New Summary: Maternal infection during pregnancy is an established risk factor for neurodevelopmental disorders, including Autism Spectrum Disorders (ASD), yet little is known about how immune insults alter neural circuitry in the developing brain and, as a result, impair behavior. One potential site for immune effects is the medial prefrontal cortex (mPFC), which plays a critical role in the higher-order social and cognitive functions compromised in disorders of altered neural development. Through the support of an exploratory R21 grant, we used a mouse model of Maternal Immune Activation (MIA) and developed a silicon probe-based multichannel recording system for high-throughput functional analysis of mPFC circuitry. Using this approach, we examined mPFC in adult offspring following maternal exposure to a viral mimetic polyinosinic:polycytidylic acid (poly(I:C)), and identified an unexpected hypofunction in the output fibers of layer 5 projection neurons as the central defect in the mPFC. In particular, layer 5 axons were less able to sustain output during prolonged activity, and their temporal precision was impaired. Transcriptomic profiling of the mPFC revealed a downregulation of the cell adhesion molecule L1cam, a putative regulator of axonal excitability. Here the proposed work will use the same mouse MIA model to determine the functional ramifications of axonal hypoactivity on specific long-range targets of the mPFC that mediate behaviors dysregulated in ASD and related disorders ? mediodorsal thalamus (MD), basolateral amygdala (BLA), and nucleus accumbens (NAc). Aim 1 of this proposal will test whether direct recruitment and/or indirect feedforward inhibition of principal cells in these subcortical regions is impaired in offspring exposed to maternal immune activation (MIA). Layer 5 mPFC projections will be specifically targeted for optogenetic stimulation with viral approaches. In Aim 2, we will augment layer 5 output with complementary genetic (L1cam rescue) or optogenetic (SSFO-driven enhanced excitability) approaches to determine whether this restores L5 output and rescues ASD-related deficits. In Aim 3, we will test for generality of our findings in MIA models and cortical regions. First, we will test whether MIA induced by lipo-polysaccharide (LPS) has similar effects as poly(I:C) on mPFC axonal function. Next we will determine whether somatosensory cortex, another cortical region implicated in ASD behaviors, also shows specific MIA induced changes in layer 5 axonal function, as might be expected if midgestational MIA broadly affects layer 5 cortical neurons at a critical step in their development. The results of the proposed experiments could both help to explain pathogenesis of ASD and other neurodevelopmental disorders and identify a molecular pathway that could be targeted to restore behavior.

PROJECT SUMMARY The CLC chloride channel family is a class of membrane proteins that controls the flux of chloride ions across cell membranes. Nine unique CLC homologs are differentially expressed in mammalian tissue and function in diverse physiological roles, ranging from electrical excitation of muscles and neurons to regulation of electrolyte balance. One subtype, CLC-2, is a voltage- dependent channel expressed broadly in the brain. Although the presence of CLC-2 in the brain has been known for decades, the role of this CLC homolog in neuronal signaling and proper brain function remains poorly understood, in part due to the absence of potent and selective small-molecule tools that enable studies of the molecular physiology of this channel. A recent breakthrough in our laboratories now opens the door to developing small molecule tools specific to CLC-2. Through a compound-library screen, we identified `hit' compounds that inhibit CLC-2 activity. We developed one of these into a potent and selective CLC-2 inhibitor, FA44, which has an IC50 of 18 nM for CLC-2 and no off-target effects on the closest CLC homolog or on a panel of 65 CNS channels, receptors, and transporters. The efficacy and selectivity of FA44 for CLC-2 is further supported by our electrophysiological recordings of brain slices from wild-type versus CLC-2 knock-out mice. In this project, we will continue our collaborative efforts to develop, characterize, and use chemical tool compounds for studying CLC-2. In Aim 1, we will identify the mechanism of action and molecular determinants for inhibition of CLC-2. In Aim 2, we will develop novel probes, including small-molecule activators and fluorescent imaging probes for localizing channel expression. In Aim 3, we will leverage our tool compounds to query the role of CLC-2 in excitatory synaptic transmission and network excitability in the thalamus and to evaluate the potential causative link between CLC-2 malfunction and epilepsy. Our team's combined expertise in synthetic chemistry (Du Bois), ion-channel structure-function (Maduke), computation (Dror), and cellular neuroscience/epilepsy (Huguenard) ideally positions us to advance this research program.

Epileptic seizures are unpredictable events that significantly reduce quality of life. Predicting when the next seizure would occur could both prepare persons with epilepsy and their caregivers, and potentially aid in the treatment of seizures. Animal models of epilepsy provide an opportunity to explore the nature of brain activity in the period leading up to seizures. Using both mouse and rat models of generalized absence epilepsy, we have found a specific build up of thalamic neural spiking activity for several seconds before each seizure. This novel electrophysiological signature occurs in the absence of any overt epileptiform EEG activity. We propose to identify the neural circuits that are responsible for pre-seizure activity using high-density multi-channel silicon probes to record broadly across seizure-generating networks in the mouse. We will also measure calcium ion levels, a readout of neural activity, in neuronal cell bodies and their output axons using fluorescent calcium indicators (GCaMPs) and multiphoton microscopy to capture a highly complementary component of pre-seizure activity with high spatial resolution. Neural activity data will be collected together with EEG, locomotion signals, sensory-evoked responses, and pupil diameter to create a comprehensive multimodal stream of pre-seizure activity. This information will be fed into unbiased machine learning approaches to develop predictive algorithms. We will directly test coupling strength within thalamocortical pre-seizure networks by conducting network-level and targeted single-cell recordings in acute brain slices. To determine a specific role of pre- seizure networks in generating seizures, we will test whether chemogenetic or optogenetic silencing of key pre-seizure network elements reduces seizure incidence or severity. Finally, we will test whether we can use seizure-predictive signals to intervene in real-time and prevent seizures before they take hold. Together, these experiments will provide proof of concept for a novel therapeutic approach: targeting the pre-seizure state to improve seizure control.