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: (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.

[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): 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.

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

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): 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.

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. !

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.