Hal Blumenfeld - US grants

The cerebral cortex has the ability to generate rhythmic synchronized network activity both in the form of normal neural rhythms as well as during epileptic seizures. A key cell type in the generation of these patterns of synchronized activity is burst generating neurons. Intrinsically bursting pyramidal cells with extensive local axon collaterals have been described previously in layer V, and more recently n layers II-III. Recent work has shown that a class of these neurons, called chattering cells, have the ability to generate sustained high frequency (30-70 Hz) burst firing. However, the mechanisms by which these cells generate burst discharges, or the functional influence of these burst discharges on postsynaptic neurons, is not presently known. Preliminary studies have shown that chattering cells have a prominent single spike afterdepolarization (ADP), and each burst is followed by a large afterhyperpolarization (AHP). In addition, some non-chattering cells can be induced to chatter by addition of cholinergic or metabotropic glutamate receptor agonists, or by prolonged depolarization. In this project we intend to use intracellular recording in an in vitro slice preparation of ferret visual cortex with the following specific aims: 1. To investigate the ionic mechanisms of the spike ADP in chattering cells, and its role in generating high frequency burst firing. 2. To investigate the ionic mechanisms of the post-burst AHP in chattering cells, and its role in determining the frequency of burst generation. 3. To investigate the mechanisms for inducing non-chattering cells with neuromodulators or depolarization to fire repetitive high frequency bursts. 4. To use dual cell recording to study the effects of high frequency burst firing on synaptic transmission. The information provided by these studies may yield important insights into the mechanisms of normal cortical rhythmic oscillations, and suggest new therapies for the therapeutic management of epileptic seizures.

[unreadable] DESCRIPTION (provided by applicant): Absence seizures occur most commonly in children as staring spells lasting 5-10 seconds, associated with a rhythmic "spike-wave" discharge (SWD) on electroencephalography (EEG). Although considered a form of generalized epilepsy, both human and animal EEG recordings suggest that SWD involve selective cortical and subcortical networks, while sparing other regions. Our central hypothesis is that increased neuronal firing in specific networks leads to regional heterogeneity in SWD. Since this may have important therapeutic significance, our main goal is to determine which specific anatomical networks are selectively involved, and whether neuronal activity during SWD increases or decreases in these brain regions. Previous studies have failed to adequately address this problem. While functional MRI (fMRI) has become a common tool in neuroscience research, here we will combine fMRI with other neuroimaging and local physiology methods to enhance data interpretation. Using an established rodent epilepsy model, we will first relate neuronal activity to neuroimaging signals through simultaneous, co-localized electrophysiology and fiber optic cerebral blood flow (CBF) and pO2 recordings during SWD. We recently calibrated blood oxygen level dependent (BOLD) fMRt; separately measuring fMRI, CBF and cerebral blood volume (CBV) to obtain quantitative maps of the cerebral metabolic rate of oxygen (CMRO2), a more direct measure of neuronal activity. Therefore, we will next obtain high spatial resolution CMRO2 maps during SWD through calibrated BOLD. Finally, we will study dynamic changes through high temporal resolution EEG-triggered fMRI during SWD. Knowledge of the specific regional networks involved in spike-wave seizures, and whether increases or decreases in neuronal activity occur, may lead ultimately to targeted treatment in these regions including gene therapy, selective )harmacological agents, or deep brain stimulation. The approach used in this well-characterized model may also enhance the ability to interpret noninvasive epilepsy neuroimaging studies in humans. [unreadable] [unreadable]

Seizures have both transient and enduring effects on nervous system function. In childhood absence epilepsy (CAE) seizures consist of brief 5-10 s episodes of unresponsiveness, associated with a 3-4 Hz spike-wave discharge (SWD) on electroencephalography (EEG). CAE affects 10-15% of children with epilepsy. In addition to transient attention deficits during seizures, many children also suffer from milder chronic deficits in attention between absence episodes. An important feature of attention deficits in CAE is that they vary in severity from one seizure to the next, and from one patient to the next. The fundamental mechanisms of variable impaired attention during and between absence seizures are not known. Functional magnetic resonance imaging (fMRI) has elucidated brain areas that normally participate in attention, including the anterior cingulate/medial frontal cortex, thalamus and other regions. Recent investigations suggest that absence seizures, long considered a generalized form of epilepsy, in fact preferentially involve specific brain networks while sparing others. Therefore, our central hypothesis is that absence seizures disrupt function in localized attention networks, leading to deficits both during and between seizures. Repeated severe impairment during seizures may be related to chronic impairment between seizures. We plan to investigate the underlying neural basis of fluctuations in attention in CAE through simultaneous behavioral, EEG, and fMRI recordings. Our first aim will be to characterize behavior during seizures using two tasks of sustained performance over time, which require different levels of attentional vigilance. Task performance will be used to categorize seizures as being associated with good or impaired performance, and we will correlate the severity of deficits during seizures with chronic deficits between seizures. Our second aim is to relate EEG changes with the variable task performance found in Aim 1. EEG power at the 3-4 Hz seizure frequency, and gamma frequency signals previously associated with attention, will be analyzed in frontal attention networks and correlated with task performance. Our third aim is to use fMRI during absence seizures to identify changes in specific attention areas such as the anterior cingulate/medial frontal cortex, thalamus, or other regions related to impaired performance. We will also relate the severity of fMRI changes during seizures to fMRI measures of chronic network dysfunction between seizures, using methods such as resting functional connectivity analysis. Understanding the fundamental mechanisms of variable impaired attention in CAE may lead to improved, targeted therapies to prevent both the transient impairments during seizures, and chronic persistent deficits.

Applications of magnetic resonance (MR) techniques to research in the life sciences are growing rapidly. State-of-the-art heteronuclear MR spectroscopy (MRS) and multi-modal MR imaging (MRI) methodologies cultivated at the Magnetic Resonance Research Center (MRRC) at Yale have moved in vivo animal research into central roles in experimental neuroscience, addressing fundamental issues with far reaching implications for brain function. Since the formation of the Yale MRRC in the early in 1980s, the number of . horizontal-bore magnets for in vivo studies have multiplied three-fold in 2004. The present number of ' magnets for in vivo studies - three each for animals and humans - were needed to match the growing number of investigators across many disciplines - a majority of whom are supported by NINDS. The strength of the Yale MRRC has been, and still is, the dynamic interaction between rodent and human research. Active interplay between heteronuclear MRS and multi-modal MRI methods in rodents and humans have furthermore rapidly progressed. Because MR technology requires unwavering infrastructural support for state-of-the-art exploits to be successfully applied, long-term stability of Yale MRRC is contingent on sustained support. A program in Quantitative Neuroscience with Magnetic Resonance (QNMR) at Yale will support shared resources and facilities used by NINDS-funded investigators at Yale, and thereby generate greater productivity than would be possible via independent efforts. QNMR will consist of three research Cores - each dedicated to improving effectiveness of ongoing research based upon multimodal MRI, heteronuclear MRS, neurophysiology - and one service Core - designed for rapid data analysis, access, sharing, and backup using high-performance cluster of workstations. We expect that QNMR will promote a more cooperative and interactive research environment for neuroscientists who are utilizing MR technology at Yale, and will nurture new cross-disciplinary approaches in medicine, physiology, and neuroscience.

In normal development there are critical periods during which learning and plasticity are enhanced. We recently found that treatment of a rodent epilepsy model with anti-seizure medication early in life, led to long-term suppression of spike-wave seizures in adulthood even after the medication was stopped. Our previous work in this rodent model demonstrated that spike-wave seizures are associated with abnormal function and structure in specific corticothalamic networks, and that these abnormalities are not present early in life before the development of seizures. Based on this, we now hypothesize that treatment early in development suppresses spike-wave epileptogenesis, and can prevent the long-term abnormalities in brain structure and function in this disorder. To translate this work into the human arena, it will be crucial to identify safe noninvasive methods to monitor biomarkers of epilepsy development in children and its prevention by therapy. Powerful neuroimaging methods now enable the noninvasive assessment of brain function and structure. Our preliminary studies have found abnormally increased resting functional connectivity on fMRI, and abnormally reduced white matter fractional anisotropy on diffusion tensor imaging (DTI) in the rodent spike-wave epilepsy model. Therefore, our aims are now to investigate fMRI resting functional connectivity and DTI as promising biomarker of epileptogenesis and its prevention by therapy. Will performing measurements of fMRI resting functional connectivity at different developmental stages in treated vs. untreated animals. We will also relate these measurements to connectivity evaluated through coherence analysis of electroencephalography. In addition, we will investigate DTI as another promising biomarker by again performing measurements at different ages in treated vs. untreated animals. We will also investigate the anatomical basis of white matter DTI abnormalities through electron microscopy to determine changes in axons and myelin in affected regions. 2.

PROJECT SUMMARY / ABSTRACT Seizures have both local and remote effects on nervous system function. Temporal lobe epilepsy is a common and debilitating neurological disorder, characterized by focal seizures arising from limbic structures, including the hippocampus. Interestingly, focal temporal lobe seizures often cause functional deficits such as impaired consciousness, which is not expected from local hippocampal impairment alone. Human focal temporal lobe seizures with impaired consciousness show slow waves on electro-encephalography (EEG) and decreased cerebral blood flow in the neocortex, distant from the hippocampus. The mechanisms by which focal seizures in the hippocampus cause depressed function in the neocortex are not known. We established a rat model with focal limbic seizures exhibiting high frequency discharges in the hippocampus, but slow 1-3 Hz activity in the neocortex, decreased cortical blood flow and metabolism, as well as decreased behavioral responsiveness resembling the human disorder. In this model we found that important subcortical arousal systems including brainstem and basal forebrain cholinergic neurons are depressed. These finding suggest that sleep-like cortical slow waves may occur in focal limbic seizures because of decreased subcortical arousal. In addition we found that putative descending GABAergic systems including the lateral septum and anterior hypothalamus are strongly activated by focal limbic seizures. Based on these findings, our central hypothesis is that focal limbic seizures activate inhibitory systems which depress subcortical arousal leading to sleep-like cortical slow waves and impaired consciousness. We plan to investigate this hypothesis at the level of neurons, networks, and behavior in a rodent model. Recent work has also raised the exciting prospect of restoring subcortical arousal to improve cortical function during seizures. Therefore, our aims are to first investigate the inputs to subcortical arousal systems using whole-cell electrophysiology to determine incoming synaptic activity; and using optogenetics to selectively activate or inhibit input pathways. Second, we will determine which subcortical arousal systems are critical for depressed cortical function by electrically or optogenetically restoring outputs from these systems during focal limbic seizures, and measure effects on the cortex through electrophysiology recordings and high-field functional magnetic resonance imaging (fMRI). Third, we will examine the effects of focal limbic seizures on attention and decision-making tasks and investigate the ability of restored subcortical arousal to improve behavioral responsiveness during seizures. The integration of information across these levels will increase our understanding of abnormal long-range network changes in epilepsy, potentially leading to new therapeutic options in the treatment of this disorder.

DESCRIPTION (provided by applicant): Impaired consciousness during epileptic seizures has serious consequences, including hazardous driving, decreased work/school performance and social stigmatization. Patients with medically and surgically refractory epilepsy often suffer from partial seizures with impaired consciousness, and no viable treatment options. Recent neuroimaging and electrophysiology findings suggest that impaired consciousness in partial seizures is related to depressed cortical function in widespread regions remote from the seizure focus. We have developed a rat model that replicates the human findings, including slow waves on electro- encephalography (EEG), decreased cerebral blood flow (CBF) and decreased functional MRI (fMRI) signals in the neocortex, as well as behavioral arrest during seizures. Advances based on both human work and the rat model demonstrates that focal seizures decrease activity in subcortical arousal structures including the upper brainstem and intralaminar thalamus. This in turn leads to sleep- or coma-like changes in the association cortex, and to loss of consciousness during and following seizures. In other disorders of consciousness, such as minimally conscious state, recent work has shown that thalamic stimulation can increase behavioral arousal. These finding pave the way for a new therapeutic approach to directly treat impaired consciousness in partial seizures: reactivation of the subcortical arousal systems through deep brain stimulation. The goal of the current proposal is to test this approach in the rat model, providing proof-of-principle efficacy for possible human therapeutic trials. Our preliminary studies suggest that stimulation of the rostral thalamic intralaminar centro-lateral nucleus (CL) converts slow wave activity in the cortex into an awake EEG pattern, and also increases cortical function based on fMRI. Therefore our aims are to first test CL thalamic stimulation under general anesthesia, to emulate the conditions during surgical device implantation. We will obtain suitable therapeutic stimulus parameters aimed to achieve cortical activation based on electrophysiology and intraoperative fMRI. Second, we will test the therapeutic efficacy of CL stimulation to improve cortical function and behavioral responsiveness during seizures in awake, behaving animals. We will test the effects of stimulation during both evoked seizures, and in response to spontaneous seizures-automatically detected using closed-loop stimulation similar to that employed in human devices. Although stopping seizures is ideal, for patients in whom seizures cannot be stopped, preventing impaired consciousness would greatly improve quality of life. If deep brain stimulation can improve cortical function and consciousness during partial seizures in an animal model, this may rapidly lead to translation of this new approach to human treatment trials.

PROJECT SUMMARY / ABSTRACT Seizures have both local and remote effects on nervous system function. Focal seizures can propagate from the site of onset to engage a larger network and induce severe consequences including impaired arousal, cardiorespiratory changes and in some cases death. Impaired cardiorespiratory function during and following seizures may contribute to chronic hypoxic brain damage and to long-term deficits in epilepsy. In addition, sudden unexpected death in epilepsy (SUDEP) is thought to arise from a state in the post-ictal period in which cardiovascular, breathing and arousal functions are impaired. Although cardiac, autonomic, breathing and arousal effects of seizures have long been recognized, the specific mechanisms by which seizures cause these changes have been relatively neglected. Our prior work investigating impaired consciousness in epilepsy suggests that seizures can depress subcortical arousal circuits including the upper brainstem. Human and animal model studies led to the network inhibition hypothesis, in which seizures inhibit brainstem arousal during and following seizures. Our preliminary data extend this work, showing that decreased ictal and post- ictal cardiorespiratory function in a rodent model is associated with markedly suppressed firing of medullary serotonergic neurons in the lower brainstem. Based on these findings, our central hypothesis is that seizures propagate to inhibitory circuits which depress lower brainstem neuromodulatory and control systems; this in turn impairs cardiorespiratory functions significant for seizure morbidity and mortality. We plan to investigate this hypothesis in detail through a combination of neuroimaging, electrophysiology, and neurotransmitter studies in a rodent model. Our aims are to first define the network of cortical and subcortical structures which cause impaired cardiorespiratory function during and following seizures using fMRI, local field and multiunit recordings, local electrical and optogenetic stimulation and inactivation experiments. Next, we will investigate the neurotransmitters producing impaired cardiorespiratory function using in vivo biosensor probe measurements. Finally, we will determine the changes in firing patterns of identified brainstem serotonergic and other modulatory neurons as well as cardiorespiratory control neurons using juxtacellular recordings during and following seizures. The integration of information across these levels will increase our understanding of abnormal long-range network changes underlying impaired ictal and post-ictal breathing and cardiac function, potentially leading to new treatment options to prevent seizure-related morbidity and mortality.

PROJECT SUMMARY / ABSTRACT Absence seizures occur most commonly in children as staring spells lasting 5-10 seconds, with rhythmic ?spike-wave? discharge (SWD) on electroencephalography (EEG). They can occur up to hundreds of times per day and are not benign, with deficits in attention and psychosocial function in some cases persisting into adulthood or even after seizure suppression. The mechanisms by which absence seizures impair cognition are not known. One intriguing but little-studied aspect of absence seizures is the fact that some episodes impair and others spare behavioral responses even within the same individual. The relationship between variable absence behavioral severity and neuronal activity may provide fundamental insights into the pathophysiology of seizures. Prior human studies and animal models have shown widespread EEG and fMRI increases as well as decreases during absence seizures. We recently found in a large patient sample that absence seizures with more severely impaired behavior had larger fMRI and EEG amplitude in widespread brain networks. We also found that abnormally enhanced fMRI synchrony persists between bilateral cortical regions even when seizures are not present in patients and in animal absence models. The neuronal basis for these changes both at rest and during SWD is not known, but our recent work in rodent absence models and normal conditions suggests that the amplitude of fMRI signals is related to changes in the total population activity of neurons. Therefore our central hypothesis is that the severity of absence seizures is determined by a combination of the number of neurons involved and their firing pattern in widespread brain networks before and during seizures. An important limitation of previous work has been anesthetic agents, which markedly alter fMRI responses and the excitability of neurons. This may explain why most animal models show cortical fMRI increases during SWD whereas human studies show a predominance of sustained cortical fMRI decreases. Because of this discrepancy the neuronal basis of physiology changes in absence seizures remains uncertain. We recently habituated genetic absence epilepsy rats of Strasbourg (GAERS) to allow awake-head fixed experiments. Initial measurements in this new model show sustained cortical fMRI and CBF decreases during SWD much more closely resembling humans. We now plan complementary high spatiotemporal resolution experiments in this awake model including fMRI, electrophysiology and behavior to fully understand the neuronal basis of variable severity in absence seizures. Our aims are to first image the networks involved at baseline and during severe versus mild SWD, and to relate the neuroimaging changes to spike and wave amplitude on EEG. Second, we will use multiunit and neuronal ensemble recordings to determine the neuronal basis of severe versus mild SWD. Third, we will relate the physiological severity of SWD to behavior through auditory detection and response tasks. The integration of information across these levels will increase our understanding of neuronal changes in absence epilepsy potentially leading to new treatment options.

PROJECT SUMMARY / ABSTRACT Impaired consciousness during seizures has a major negative impact on quality of life for people with epilepsy. Consequences include risk of motor vehicle accidents, drowning, poor work and school performance, and social stigmatization. Impaired ictal/postictal arousal may also compromise breathing leading to sudden unexpected death in epilepsy. Although the primary goal of epilepsy care is to stop seizures, restoring conscious awareness in patients whose seizures cannot be stopped (by medications, surgery or deep brain stimulation) could significantly improve outcome. Disorders of consciousness other than epilepsy have long been known to arise from dysfunction of subcortical-cortical arousal circuits. Deep brain stimulation (DBS) of the thalamic intralaminar central lateral nuclei (CL) is a promising approach to restore conscious arousal currently being trialed for chronic disorders of consciousness (N. Schiff, NINDS UH3 NS095554). Recent neuroimaging and EEG studies have shown that transient impaired consciousness in temporal lobe epilepsy (TLE) seizures also depends on subcortical-cortical arousal including thalamic CL. Translational studies from our research group further demonstrate depressed CL function in limbic seizures, and most importantly that thalamic CL stimulation has the potential to restore physiological and behavioral arousal in the ictal and postictal periods. DBS treatment of epilepsy has advanced rapidly with FDA approval of responsive neurostimulation (RNS, NeuroPace) and thalamic anterior nucleus stimulation (Medtronic). Investigational devices such as the RC+S (Medtronic) provide a unique opportunity for responsive stimulation of up to 4 separate brain regions, enabling conventional sites such as hippocampus (HC) to be combined with innovative targets such as thalamic CL. Meanwhile, Dr. Worrell?s group at Mayo has developed the Epilepsy Personal Assistant Device (EPAD), a custom application running on a hand-held device with bi-directional communication with the RC+S. The EPAD will enable cloud-based data storage, seizure diaries, and automatic behavioral tests similar to those we have validated previously. Therefore, our goal is to develop and pilot test the feasibility and safety of bilateral thalamic CL stimulation using RC+S to restore conscious arousal in TLE seizures which are not stopped by conventional responsive neurostimulation, offering hope to greatly improve quality of life in these patients. Our aims are to first conduct final benchtop preclinical verification of RC+S and EPAD algorithms for CL stimulation leading to FDA IDE approval. Second, we will initiate a small clinical trial implanting RC+S in patients with refractory TLE and beginning with open-label HC stimulation and baseline EPAD behavioral testing. Third, we will adjust responsive thalamic CL stimulation parameters for arousal. Finally, we will test safety and initial feasibility of responsive CL stimulation to restore arousal during seizures.