Dennis D. Spencer - US grants

This program project focuses research clinicians and basic neurobiology investigators on the analysis and modeling of human epileptogenic tissue. The surgically resected tissue will come from two well-defined human seizure disorders, temporal lobe epilepsy associated with medial temporal sclerosis (medial temporal lobe epilepsy (MTLE), and epilepsy associated with chronic tumors (tumor associated epilepsy (TAE). Experimental neurobiological techniques will be used to address specific hypotheses relating to the electrophysiology, anatomy, and biochemistry of the hippocampus and parahippocampus in MTLE and of the tumor with surrounding cortex in TAE. The program is divided into three major sections. The first defines the tissues' electrophysiological characteristics using depth electrodes and intraoperative recording and stimulating techniques (Project 1, S. Spencer). This same tissue is then studied in slice preparations asking similar questions at an intracellular and network level (Project 2, A. Williamson). The second section uses immunohistochemical, autoradiographical, and molecular techniques to characterize structure (Project 3, N. de Lanerolle). Data from all three projects is correlated and compared with a heat-kindled rat model (Project 3, N. de Lanerolle). The third section studies the neurobiology of ion-regulating proteins in the tissue of these two human focal epileptic conditions attempting to understand what cellular (particularly glial) events predispose to neuronal death and resultant excitability and more specifically, Dr. A. Cornell-Bell (Project 5) will characterize glial Ca++ physiology and glutamate pharmacology in human and animal tissue using optical imaging techniques.

This program project focuses research clinicians and basic neurobiology investigators on the analysis and modeling of human epileptogenic tissue. The surgically resected tissue will come from two well-defined human seizure disorders, temporal lobe epilepsy associated with medial temporal sclerosis (medial temporal lobe epilepsy (MTLE), and epilepsy associated with chronic tumors (tumor associated epilepsy (TAE). Experimental neurobiological techniques will be used to address specific hypotheses relating to the electrophysiology, anatomy, and biochemistry of the hippocampus and parahippocampus in MTLE and of the tumor with surrounding cortex in TAE. The program is divided into three major sections. The first defines the tissues' electrophysiological characteristics using depth electrodes and intraoperative recording and stimulating techniques (Project 1, S. Spencer). This same tissue is then studied in slice preparations asking similar questions at an intracellular and network level (Project 2, A. Williamson). The second section uses immunohistochemical, autoradiographical, and molecular techniques to characterize structure (Project 3, N. de Lanerolle). Data from all three projects is correlated and compared with a heat-kindled rat model (Project 3, N. de Lanerolle). The third section studies the neurobiology of ion-regulating proteins in the tissue of these two human focal epileptic conditions attempting to understand what cellular (particularly glial) events predispose to neuronal death and resultant excitability and more specifically, Dr. A. Cornell-Bell (Project 5) will characterize glial Ca++ physiology and glutamate pharmacology in human and animal tissue using optical imaging techniques.

Epilepsy is the most common chronic neurological disease, affecting almost 1% of humans from infancy to old age. It exists in a variety of forms and whatever initiates a seizure, allows its propagation and causes cessation of the event is not completely understood. What is understood is the serious impact this disease has on the medical, social, and economic well being of the patient. However, many remarkable new insights into epilepsy have accompanied the rapid growth of Magnetic Resonance (MR) technology. The instruments and the computer driven manipulation of both the hardware and the post acquisition data have provided tools that can solve many of epilepsy's mysteries given the proper dialogue between physicists and clinicians. These collaborations have already provided superior anatomical images and have allowed reclassification of some disease processes. Precise localization of biochemical alterations in epilepsy.is a reality using different.modes of spectroscopy, and fast MR will allow us to noninvasively image function of the brain including subtle cognitive processes. The purpose of this grant is to support a 2.5 day invited interdisciplinary meeting of 70 international MR physics experts and epilepsy clinicians who are active in MR collaborative research. They will exchange information and research questions in an informal format of tutorials, brief presentations and open discussions. This is meant to foster collaborations and new research initiatives both within and between centers. The meeting will guide the physicists towards the most relevant disease questions and broaden the epileptologists' appreciation and utilization of this rapidly advancing technology. An attempt will be made to promptly publish the output of this dialogue.

neurotransmitter transport; neuropharmacology; anticonvulsants; brain metabolism; gamma aminobutyrate; partial seizure; sodium ion; calcium ion; pharmacokinetics; exocytosis; membrane transport proteins; clinical research; human subject; microdialysis;

The Core provides resources that are either directly shared by two or more projects or that promote project interaction. The PI is responsible for the Core and for assessing how each project interacts with the others. Glutamate clearance and energy metabolism derangement must be put into the context of patient selection, conduct of operative intervention, uniform description of tissue to be studied, and outcome regarding seizure control. The core will, therefore, be divided into four sections: Section 1: Preoperative definition of the patient population and presumed epileptogenic substrate. In this section, the PI will first define the patients that will undergo study, will obtain informed consent, will place electrodes in those patients requiring invasive study, and will make sure that all preoperative and qualitative and quantitative imaging and electrophysiology has been carried out (patient selection at conference, MRI, volumetrics, AVEEG, PET, MRS). Microdialysis assessment of glutamate will be provided both in vivo, when patients undergo invasive study, and in the slice. This data will be used correlatively in all the projects. Section: Intraoperative 13 study and tissue distribution. This involves digitized imaging of the brain intraoperatively to document the site of electrophysiological abnormalities and hippocampus in photographed, sectioned and numbered for investigator review. Section 3: Postoperative tissue characterization. This section is devoted to the neuronal and glial cell counts performed by Dr. Jung Kim which provides the cellular substrate for the metabolic and energy correlation studies. Section 4: Project coordination. The PI will provide coordination and data sharing among the projects assisted by an administrative associate who will maintain weekly conferences, coordinate patient movement among all the preoperative tests including the trips to Brookhaven National Laboratories. The administrator is also responsible for collecting and collating all data, coordinating interdisciplinary studies, and scheduling internal and external advisory meetings.

A variety of metabolic imaging studies, including FDG PET and MRS, have suggested metabolic and energy deficiencies in epileptogenic regions of certain symptomatic epilepsies. In particular, the 1H NMR measurements of N-acetyl aspartate (NAA) may indicate mitochondrial dysfunction and the 31P NMR demonstrates depleted high energy phosphates. The medial temporal lobe epilepsy (MTLE) patients have been the most frequently studied and localized by these methods. Hippocampal tissue removed from these patients to control medically intractable seizures has revealed cytochrome c oxidase (COX) and Na/k ATPase dysregulation also suggesting that the metabolic imaging studies may be detecting mitochondrial dysfunction or loss. In vivo hippocampal microdialysis studies have implicated glutamate transporter malfunction as a significant function link tying a low energy state to poor glutamate clearance and excess excitation. Thus, the hypothesis of this grant states that energy compromise may exist in MTLE triggering an excitatory cascade with inadequate glutamate metabolism and altered neuronal-glial neurotransmitter cycling and subsequent poor extracellular glutamate clearance leading to the spread of excitability. Project 1 will initially address this hypothesis by examining CMR glucose with FDG PET, high energy phosphates (PCr and ATP) with 31 PNMR and a neuronal dysfunction with 1H NMR of NAA. These measurements of tricarboxylic acid (TCA) 13C and anaplerotic rates of glutamate cycling and glutamine synthesis will be addressed in vivo with controls and correlated with in vitro 13C cycling rates in the resected tissue described in Project 2. Project 3 will correlate these findings with human tissue mitochondrial dysfunction, density, hyperplasia and DNA analysis along with COX and glutamate transporters. Finally, the functional consequences of this hypothesis will be tested in Project 4 examining the ECF space, k+ and glutamate regulation in the human and control kainate rat model.

Subcortical white matter (SCWM) neurons are a common feature in the primate neocortex and are present in large numbers in the human. These cells are believed to be the remnant of the subplate and may have served guidepost functions during neocortical development. The function of SCWM neurons in the adult is unknown, however because they have extensive axonal processes in early postnatal life, they may be able to affect the activity of large regions of cortex into adulthood. Although the significance is not yet known, there are also increased numbers of SCWM neurons in a variety of neurological diseases, including schizophrenia, forms of epilepsy and, perhaps, Alzheimer's disease. It is important, therefore, to examine the synaptic organization and physiology of these cells for a complete understanding of both normal and pathological cortical function. The goal of this project is to test the hypothesis that SCWM neurons are integrated into the neocortical circuitry. This study is uniquely suited to be carried out using human tissue for two reasons; 1) the density of SCWM neurons increases with the complexity of the organism, therefore studies carried out in animals such as rodents or cats may not be applicable to humans; 2) the costs of carrying out such a study in non-human primates would be prohibitive. Tissue from patients undergoing resection for the treatment of intractable epilepsy is routinely available. These resections typically include areas involved in seizure generation as well as relatively normal tissue adjacent to these areas. We will examine whether SCWM neurons are integrated into the neuronal circuitry using a combination of anatomical and physiological techniques. If this hypothesis is valid, there should be evidence for both synaptic inputs and outputs between these cells and the neocortex. Electron microscopic studies of this tissue will be performed to examine whether there are synapses onto the soma and proximal dendrites of these cells that arise from the cortex. In addition, the synaptic output of these cells will be assayed by examining the axonal arbors of biocytin- filled cells. The synaptic targets of SCWM cells will be identified using double labeling studies. Physiological studies will be performed by recording from visually identified SCWM neurons. In addition to characterizing these cells physiologically, we will examine whether these cells receive spontaneous or evoked synaptic activity from the neocortex and the possible transmitter(s) underlying any synaptic activity. All cells studied physiologically will be labeled with biocytin to allow us to verify the cell type and for use in the anatomical experiments. These studies will provide the first information on the possible role of these cells in the function of the adult neocortex. Given the possible involvement of these cells in a variety of neurological disorders, it is critical to characterize these cells completely.

clinical research; human subject;

[unreadable] DESCRIPTION (provided by applicant): Extracellular glutamate is known to be excessively elevated both ictally and interictally in the epileptic human hippocampus. However, the source and significance to epileptogenesis of these high levels of glutamate remain a matter of debate. The overall goal of this proposal is to better understand the cellular and metabolic mechanisms that underlie the elevations of glutamate seen in the hippocampus of patients with mesial temporal sclerosis (MTS). There is compelling evidence for both neuronal and glial dysfunction n MTS and much of the work to date has focused on changes in either neuronal or glial function in isolated preparations. We postulate that the "neuronal-glial unit" is impaired in MTS such that the dysfunctional glia Droduce the bulk of the elevated interictal glutamate. Moreover, while both neuronal and glial release may contribute to ictal elevation, we hypothesize that the glial component is very substantial. We propose to test this hypothesis using a wide array of techniques aimed at understanding the disposition of glutamate in the epileptogenic human temporal lobe. We will couple these studies to the intracranial monitoring used to dentify epileptogenic regions in patients with medically intractable epilepsy. In Specific Aim 1, we will use HPLC to measure extracellular glutamate and glutamine in microdialysate both interictally and following stimulation using microdialysis probes attached to the depth electrodes. Stimulation will be done to simulate focal seizure activity. In Specific Aim 2, we will infuse 13C acetate, which is exclusively taken up by glia, and will use mass spectrometry to measure the fractional enrichment of 13C in glutamate and glutamine in the microdialysate at baseline and following stimulation to determine the source(s) of extracellular glutamate under these different conditions. For both these studies, we will perform the analyses in probes placed in the hippocampus and in a non-epileptogenic cortical region. Glutamate uptake and efflux will be measured in Specific Aim 3 using resected cortical and hippocampal tissue. In addition physiological studies on the effects of elevated glial glutamate will be done as part of this aim. Possible mechanisms for glial release will be addressed as well. Finally, in Specific Aim 4, the levels of the enzymes and transporters most relevant to glutamate metabolism in glia and neurons will be measured by Western blots using resected tissue. The data obtained in each Specific Aim will be correlated with quantitative neuronal and glial counts. Relevance: Taken together these data will allow us to develop a better understanding of the origins of the elevated glutamate in MTS, and thus to devise more targeted molecular and cellular therapies to restore glutamate homeostasis and thus decrease excitability and seizures. [unreadable] [unreadable] [unreadable]