Paul S. Buckmaster - US grants

The applicant's broad, long-term objective is to better understand the pathophysiological mechanisms of epilepsy, so that epileptic patients (in human and veterinary medicine) can be diagnosed and treated more effectively. The goal of the proposed project is to investigate the mechanisms underlying temporal lobe epilepsy by studying the hippocampus of the kainic acid-treated rat, which is a widely accepted model of temporal lobe epilepsy. The general hypotheses of this project are that kainic acid-treatment causes reduced excitatory synaptic drive to inhibitory interneurons, resulting in less inhibition of CA1 pyramidal cells, and that kainic acid-treatment causes reorganization of granule cell and CA 1 pyramidal cell axon collaterals, leading to recurrent excitatory circuits. Furthermore, it is hypothesized that these changes produce hyperexcitability and reduce the seizure threshold in the hippocampus. It would be difficult, or impossible, to adequately address these issues using the hippocampal slice technique; therefore, an in vivo intracellular recording and labeling preparation will be used. The specific aims of this project are to compare kainic acid-treated and control rats to: (1) identify the source of regenerative afferent fibers to CA1 pyramidal cells; (2) examine the intrinsic and synaptic physiology of CA1 pyramidal cells and dentate granule cells; (3) determine whether there is long-term hyperexcitability and reduced seizure threshold that correlates with hilar neuron loss and mossy fiber sprouting in the dentate gyrus; (4) describe quantitatively the extent of mossy fiber sprouting and identify the postsynaptic targets of the axon sprouts; and (5) investigate the intrinsic and synaptic physiology and axon projections of inhibitory interneurons.

The applicant's broad, long-term objective is to better understand the pathophysiological mechanisms of epilepsy, so that epileptic patients (in human and veterinary medicine) can be diagnosed and treated more effectively. The goal of the proposed project is to investigate the mechanisms underlying temporal lobe epilepsy by studying the hippocampus of the kainic acid-treated rat, which is a widely accepted model of temporal lobe epilepsy. The general hypotheses of this project are that kainic acid-treatment causes reduced excitatory synaptic drive to inhibitory interneurons, resulting in less inhibition of CA1 pyramidal cells, and that kainic acid-treatment causes reorganization of granule cell and CA 1 pyramidal cell axon collaterals, leading to recurrent excitatory circuits. Furthermore, it is hypothesized that these changes produce hyperexcitability and reduce the seizure threshold in the hippocampus. It would be difficult, or impossible, to adequately address these issues using the hippocampal slice technique; therefore, an in vivo intracellular recording and labeling preparation will be used. The specific aims of this project are to compare kainic acid-treated and control rats to: (1) identify the source of regenerative afferent fibers to CA1 pyramidal cells; (2) examine the intrinsic and synaptic physiology of CA1 pyramidal cells and dentate granule cells; (3) determine whether there is long-term hyperexcitability and reduced seizure threshold that correlates with hilar neuron loss and mossy fiber sprouting in the dentate gyrus; (4) describe quantitatively the extent of mossy fiber sprouting and identify the postsynaptic targets of the axon sprouts; and (5) investigate the intrinsic and synaptic physiology and axon projections of inhibitory interneurons.

DESCRIPTION: (Adapted from the Investigator's Abstract): Temporal lobe epilepsy is the most common form of epilepsy in adults, and it is often medically intractable. In the dentate gyrus of patients with temporal lobe epilepsy granule cell axons reorganize. The new synaptic targets of granule cell axons must be identified. The possible targets in the dentate gyrus are other granule cells and GABAergic interneurons. Contacts with neighboring granule cells will form a recurrent excitatory circuit that could generate seizures. Contacts with GABAergic interneurons will strengthen the recurrent inhibitory circuit having an anti-epileptic effect. These alternatives are not mutually exclusive. This project will determine how the connectivity of granule cells changes in the epileptic brain. The specific aims are to define the number of synaptic contacts formed and identify the postsynaptic cells before and after kainate induced granule cell axon reorganization. Individual granule cells will be labeled intracellularly in vivo with biocytin. Within the dentate gyrus their complete axon arbors will be 3-dimensionally reconstructed and measured. Using electron microscopy the synaptic density (number of synapses per axon length) of selected axon segments will be measured. Data on axon length and synapse density will be integrated to estimate the total number and distribution of synapses made by individual granule cells. Electron microscopy and post embedding immunocytochemistry for GABA will be used to determine the proportion of synapses formed with GABAergic versus non-GABAergic neurons. These experiments will provide new data on the connectivity of granule cells and establish an experimental approach for evaluating potential treatments designed to influence granule cell axon reorganization.

DESCRIPTION (provided by applicant): Temporal lobe epilepsy is common and difficult to treat. Our long-range research goal is to help reveal mechanisms of temporal lobe epilepsy and develop anti-epileptogenic strategies. During the last funding period we discovered in a rat model of temporal lobe epilepsy that initial loss of interneurons reduces numbers of GABAergic synapses with granule cells, but over time surviving interneurons sprout axons and develop excessive synapses. However, in epileptic dentate gyrus, although GABAergic synapses are abundant, at least some (for example, basket cell-to-granule cell synapses) are dysfunctional. During the next funding period, we propose 3 specific aims. Aim 1 is to test whether activation of mTOR signaling pathway contributes to GABAergic axon sprouting after epileptogenic injuries. We will use GIN mice, which express green fluorescent protein (GFP) in a subset of somatostatin interneurons. Extent of axon sprouting will be measured by stereological analysis of GFP-immunoreactive axons, biocytin-labeling of axons of individual GFP-positive interneurons, and probability of recording GFP-interneuron-to-granule cell unitary IPSCs (uIPSCs). Aim 1 will reveal whether or not mTOR signaling pathway contributes to epilepsy-related GABAergic axon sprouting. If not, based on previous findings, it would suggest mTOR pathway activation may be specific for excitatory mossy fiber sprouting. If so, it will establish a novel and innovative method for future studies to manipulate extent of GABAergic synaptogenesis and test whether it promotes or inhibits epileptogenesis. Aim 2 is to identify mechanisms underlying increased failure rate of basket cell-to-granule cell uIPSCs after epileptogenic injuries. We will use variance-mean analysis to estimate average release probabilities and minimum numbers of release sites by recording basket cell-to-granule cell uIPSCs in slices from control and epileptic pilocarpine- treated rats. Serial-section electron microscopy will be used to measure numbers of docked vesicles at GABAergic synapses with granule cell somata in control and epileptic rats. These experiments will more precisely identify why basket cell-to-granule cell synapses are more likely to fail in epileptic tissue, which will help future attempts restore normal function at these critical synapses. Aim 3 is to test whether hilar somatostatin interneuron-to-granule cell synapses are dysfunctional like basket cell-to-granule cell synapses after epileptogenic injuries. Approaches of Aim 2 will be used. Together, proposed experiments will advance understanding of how inhibitory synaptic transmission is affected in temporal lobe epilepsy. Ultimately, we expect data generated will help advance the long-term goal of developing anti-epileptogenic treatments.

DESCRIPTION (provided by applicant): The United States needs more veterinary researchers. Veterinary researchers advance biomedical and behavioral research, support the veterinary profession, and improve the quality of animal care. This program's primary objective is to help veterinary students develop research careers. We seek highly qualified 1st and 2nd year students (eight/summer) and place them in strong research laboratories. We provide them with mentored research experience and knowledge about further research training and career opportunities. Our goal is to stimulate veterinary students'interest in research and nurture a sense of belonging to a community of veterinary scientists. The three month program consists of an individual research project, research-related workshops, career development sessions, a veterinary student journal club, and a student research symposium. The research project is the most important part of the program and is similar to a 1st year Ph.D. graduate student laboratory rotation. Participating faculty offer research opportunities in a broad range of biomedical and behavioral disciplines and are members of the following departments in the Stanford University School of Medicine: Microbiology &Immunology, Neurology &Neurological Sciences, Orthopedic Surgery, Pathology, Pediatrics, Psychiatry &Behavioral Sciences, Radiation Oncology, and Comparative Medicine. The long-term effect of this program, and others like it, is further development of the nation's workforce in laboratory animal medicine, comparative pathology, and comparative medicine.

DESCRIPTION (provided by applicant): Temporal lobe epilepsy is the most common form of epilepsy in adults and one of the most difficult types to treat. My long-range research goal is to help reveal the mechanisms of temporal lobe epilepsy so that more effective treatments and preventative strategies can be developed. It has been proposed that epileptogenic injuries kill interneurons in the dentate gyrus reducing inhibition of granule cells and lowering seizure threshold. Recent results from my laboratory provide new support for this mechanism. We propose to test the hypotheses that granule cells lose inhibitory synaptic contacts after epileptogenic injuries (Specific Aim 1) and that surviving interneurons form new inhibitory synaptic contacts with granule cells after epileptogenic injuries (Specific Aim 2). To achieve these specific aims we will estimate the total number of inhibitory synaptic contacts with granule cells at three time points during the epileptogenic process: before injury, during the latent period, and during the stage of chronic epilepsy. A newly developed stereological electron microscopic method will be used with post-embedding immunocytochemistry for gamma-aminobutyric acid (GABA) to estimate synapse numbers in control and pilocarpine-treated rats. In addition to loss of inhibitory synapses, granule cell inhibition could be reduced by decreased release of GABA from surviving interneurons. Basket cells are an important source of GABAergic synaptic input to granule cells. We propose is to test the hypothesis that basket cell-to-granule cell synapses become less effective after epileptogenic injuries (Specific Aim 3). To achieve this specific aim we will measure frequency-dependent synaptic depression at basket cell-to-granule cell synapses by recording unitary inhibitory postsynaptic currents of synaptically coupled pairs. The proposed experiments will provide new data on how inhibitory circuits in the dentate gyrus change in epileptic animals and how those changes may contribute to compensatory mechanisms and epileptogenesis.

DESCRIPTION (provided by applicant): Temporal lobe epilepsy is common and difficult to treat. Our long-range research goal is to help reveal mechanisms of temporal lobe epilepsy and develop anti-epileptogenic strategies. During the last funding period we discovered rapamycin suppresses granule cell axon (mossy fiber) sprouting and found that despite substantial reductions, spontaneous seizure frequency was unchanged in a mouse model of temporal lobe epilepsy. These findings raise doubts about the role of mossy fiber sprouting in epileptogenesis. However, scattered clues in the literature and preliminary findings suggest another recurrent, excitatory circuit in the dentate gyrus developed despite suppression of mossy fiber sprouting. The goal of the next funding period is to test the hypothesis that in a rat model of temporal lobe epilepsy surviving mossy cells function as aberrant network hubs, establish an over-developed positive-feedback circuit within the dentate gyrus, and contribute to the generation of spontaneous seizures. We propose 3 specific aims. Aim 1 is to test the hypothesis that surviving mossy cells enlarge, elongate dendritic processes, and receive more excitatory synaptic input in epileptic pilocarpine-treated rats. Hippocampal slices, whole-cell patch recording, and biocytin labeling will be used. Aim 2 is to test the hypothesis that surviving mossy cells sprout axon collaterals and form new synapses with granule cells in epileptic pilocarpine-treated rats. In vivo intracellular biocytin-labeling and electron microscopy will be used. Aim 3 is to perform 3 experiments to test the hypothesis that surviving mossy cells contribute to seizure generation. Unit recordings will be obtained from mossy cells in epileptic pilocarpine- treated rats as they experience spontaneous seizures. Seizure frequency and numbers of surviving mossy cells will be measured to determine whether there is a correlation. And, we will test whether epilepsy-related changes in mossy cell circuitry are resistant to rapamycin treatment. Achieving these aims will clarify the role of surviving hilar mossy cells in temporal lobe epilepsy. If mossy cells do play an epileptogenic role, then they may offer new therapeutic targets to dampen excitability and provide better seizure control for patients.

Project Summary/Abstract Our program provides veterinarians with rigorous research training leading to the PhD degree at Stanford University. They join top-ranked graduate home programs in the biosciences, are mentored by outstanding researchers, and develop stronger ties to their veterinary profession through the Department of Comparative Medicine. The rationale is that intense research training will enable more veterinarians to compete effectively for research grant support and become independent principal investigators, which will address a national need. Trainees choose one of 14 graduate home programs in the Biosciences: biochemistry, biology, biomedical informatics, biophysics, cancer biology, chemical & systems biology, developmental biology, genetics, immunology, microbiology & immunology, molecular & cellular physiology, neurosciences, stem cell biology & regenerative medicine, and structural biology. Program faculty include six comparative medicine mentors (who are veterinarians and faculty in the Department of Comparative Medicine), and 17 research mentors. During the next funding cycle, funds are requested to support 4 trainees at any one time. Overall, we seek to produce highly trained veterinary researchers that will assume leadership roles and exert a sustained, powerful influence in their research field and in the veterinary profession.

Our long-term goal is to help elucidate mechanisms of temporal lobe epilepsy and develop more effective treatments for patients. During the previous funding period we discovered increased activity of dentate granule cells beginning minutes before spontaneous seizures in a rat model of temporal lobe epilepsy. We now propose to further evaluate the entorhinal-hippocampal circuit to localize the site(s) of earliest preictal activity (Aim 1), test potential mechanisms of seizure initiation (Aim 2), and determine whether unit recording can be used to predict seizures in real time (Aim 3). Aim 1 is to use tetrodes to record unit activity in dentate gyrus, CA1, CAS, subiculum, and medial entorhinal cortex (layers II, III, and V/VI) in epileptic pilocarpine- treated rats as they experience spontaneous seizures. Electrographic seizure onset will be standardized by objective methods applied to field potential recordings from dentate gyrus in all cases. Neuron firing rates will be plotted with respect to seizure onset and analyzed to determine whether and when they significantly exceed baseline levels. The region(s) displaying earliest preictal unit activity will be identified as a potential site of seizure initiation. Aim 2 is to begin testing hypotheses of seizure initiation: that in epileptic rats granule cells fail to provide sufficient excitatory synaptic drive to GABAergic interneurons (Experiment 2a) and that within the aberrant, recurrent, excitatory dentate circuit granule cells with basal dendrites are hyper- connected elements and therefore potential initiators or propagators of seizure activity (Experiment 2b). In collaboration with John Huguenard we will use laser scanning photo-uncaging of glutamate to focally activate granule cells in hippocampal slices while obtaining whole cell recordings of evoked excitatory postsynaptic currents in interneurons (Experiment 2a) and granule cells (Experiment 2b), which will be labeled with biocytin to determine whether they have basal dendrites or not. Aim 3 is to develop classifier algorithms and use on-line multi-unit recording and analysis to predict spontaneous seizures of epileptic pilocarpine-treated rats in real time. Broadband recordings of prolonged periods of interictal and preictal activity will be obtained, evaluated, and made available to other investigators. The proposed experiments will help reveal underlying causes of temporal lobe epilepsy by beginning to localize the brain region(s) displaying earliest pre-seizure activity and test potential mechanisms of seizure initiation. In addition, we will attempt for the first time to predict seizures by monitoring action potential firing rates. If successful in epileptic rats, this method may eventually lead to seizure prediction devices for patients.

DESCRIPTION (provided by applicant) Harmful blooms of domoic acid-producing algae are a growing problem. Species affected include some that are threatened or endangered. Humans are at risk, and consequences can be severe. Despite established seafood testing mechanisms, concerns about potential human exposures persist, including in utero exposure, which might go undetected initially but have detrimental long-term consequences. Previous rodent studies suggest in utero exposure to domoic acid might cause neuropathological changes in the hippocampus and the development of epilepsy, but direct evidence of spontaneous seizures is lacking. The investigators will attempt to fill this critical gap. The proposed project is an interdisciplinary collaboration among wildlife veterinarians and biomedical scientists to test the hypothesis that in utero exposure to domoic acid causes temporal lobe epilepsy, albeit a milder form than that caused by adult exposure. Aim 1 is to test whether in utero exposure to domoic acid causes temporal lobe epilepsy in mice and to compare the neuropathology to that of mice exposed as adults. Aim 2 is to test the hypothesis in California sea lions by evaluating brain tissue from animals euthanized because of domoic acid toxicosis with a poor prognosis. Results will indicate whether or not in utero exposure can cause temporal lobe epilepsy to develop, and if so, whether it is as severe as that which develops after adult exposure. Findings will inform future decisions on whether and how to further evaluate risks of in utero exposure to domoic acid in humans. In addition, Aim 2 will generate rare, perfusion-fixed, quantitative whole-brain data from animals that have a large, gyrencephalic brain and naturally occurring temporal lobe epilepsy, which could provide insight into mechanisms of epileptogenesis. Public Health Relevance: The proposed project addresses growing concerns that human embryos might be at risk when pregnant women consume seafood containing naturally occurring toxins at levels below current regulatory limits. The investigators will determine whether or not exposure during pregnancy can cause temporal lobe epilepsy in laboratory mice and naturally exposed sea lions.

? DESCRIPTION (provided by applicant): Determining how spontaneous seizures initiate in patients with temporal lobe epilepsy is our long-term goal. The main hypothesis of this project is that a structural or neurochemical abnormality, usually within the ventral hippocampal formation, causes occasional, spontaneous, and progressive increases in neuronal activity, and when a threshold is passed a seizure initiates. The hypothesis predicts that a structural or neurochemical abnormality would be most severe in seizure-initiating regions that increased pre-ictal activity of principal neurons would be earliest in seizure-initiating regions, and that fcal inhibition of seizure-initiating regions would reduce seizure frequency. We propose to use epileptic pilocarpine-treated rats to test these predictions in the following aims. Specific Aim 1 s to test whether GABAergic neuron loss is most severe in seizure- initiating regions. Rats will be implanted with fine wires bilaterally in the ventral hippocampus, ventral subiculum, dorsal hippocampus, entorhinal cortex, amygdala, olfactory cortex, and septum, which are brain regions that have displayed earliest spontaneous seizure activity in the rat model. In individual subjects relative timing of electrographic onsets of spontaneous seizures will be measured to identify seizure onset site(s). In situ hybridization for glutamic acid decarboxylase and stereology will be used to quantify GABAergic neuron loss and determine whether it is more severe in seizure-initiating regions. Specific Aim 2 is to test whether action potential firing rats of principal neurons in seizure-initiating regions increase earliest before electrographic onsets o spontaneous seizures. Fine wires will be used to obtain local field potential recordings bilaterall from brain regions previously found to have earliest spontaneous seizure activity. In the same animals, tetrodes will be used to obtain single-unit recordings from principal neurons in the ventral subiculum. Spontaneous seizures will be sorted according to whether they initiated in the ventral subiculum versus elsewhere, and the timing of significantly increased average preictal firing rates will be compared. Specific Aim 3 is to test whether focal inhibition at electrographic onset sites is necessary and sufficient to block spontaneous seizures. Will inhibition of any preictally activated node in a network block ictogenesis? Or, is it necessary to inhibit the precis site of electrographic seizure onset? To address these questions, fine wires will be used to obtain local field potential recordings bilaterally from brain regions previously found to have earliest spontaneous seizure activity. A cannula will be implanted and a mini-osmotic pump will be used to continuously deliver vehicle for 1 month then tetrodotoxin for 1 month to focally inhibit activity in the ventral subiculum. Seizure frequency/severity/duration will be compared during vehicle- versus tetrodoxin-infusion. Effects of inactivating the ventral subiculum will be compared with respect to seizure onset site (ventral subiculum versus elsewhere). The proposed experiments address the most fundamental question in epilepsy research: How do spontaneous seizures start?