David A. Prince, M.D. - US grants

Electrophysiological studies will be performed on immature cortical neurons in the in vitro neocortical slice preparation, and on neurons dissociated from neocortical slices. The long term objectives of these experiments are to define the development of electrophysiological properties in immature neocortical neurons and to examine the mechanisms of epileptogenesis in the immature cortex. Specific aims include an examination of the sequence of development of subthreshold membrane properties and spike generation in immature neurons, and to examine the mechanisms of epileptogenesis in immature neocortical slices. Responses of immature slices to GABA antagonists and Ba++ will be examined at different developmental stages. Neurons will be identified directly in dissociated preparations or using intracellular HRP injections in slices. Standard current clamp techniques as well as patch clamp for whole cell recording and voltage clamp measurements will be employed. The results of these experiments will be relevant to general issues of normal and abnormal function in the immature cortex and will lead to a better understanding of the mechanisms underlying epilepsy in the immature brain.

The proposed Program uses approaches within the disciplines of neuroanatomy, neuropharmacology, and neurophysiology to study a) electrophysiological abnormalities in injured neurons and those of chronic epileptiform foci; b) the physiology of GABA-ergic neurons in cortex and thalamus; c) actions of neurotransmitters including ACh, GABA, norepinephrine, and serotonin; d) normal organization and physiology of cortical neurons and mechanisms for initiation and propagation of epileptiform discharges in cortex; e) influences of ascending cholinergic and noradrenergic systems on cortical neuronal activities and epileptiform spike-wave activities in a simple vertebrate nervous system; f) the role of serotonin subtypes and modulation of cortical excitability; and g) mechanisms of glutamate neurotoxicity on cortical neurons. The specific projects are: I. Regulation of neuronal excitability and epileptogenesis; II. Functional organization of local cortical circuits; III. Serotonin receptor subtypes and cortical excitability; IV. Subcortical influences on cortical excitability; and V. Glutamate neurotoxicity. The techniques employed include intracellular recordings; application of neurotransmitters and other agents; patch clamp recordings of whole cell currents; field potential analysis; use of cortical slice, cultured and acutely dissociated neocortical neurons and turtle nervous system in vitro; intracellular and retrograde neuronal labeling; immunocytochemistry; radioligand binding and autoradiography. The long-term goals of the Program are to obtain information which will provide a better understanding of the pathogenesis of epilepsy and insights which will lead to the development of rational new approaches to the prevention and treatment of this disorder. The studies outlined will also contribute to our understanding of normal regulation of cerebral excitability through effects of functional connections, specific transmitter systems, and intrinsic neuronal properties.

histology; biomedical facility; neocortex; experimental brain lesion; immunocytochemistry;

Clinical observations suggest that cortical malformations are an important etiological factor in the development of epilepsy, yet little is known about the mechanisms by which malformed cortex becomes prone to epileptogenesis. The planned experiments employ a model of a focal microgyrus in rat neocortex produced by a transcranial freeze lesion on the day of birth. The underlying pathology, when examined in mature animals, closely represents that of human microgyri. Electrophysiological recordings show that the area immediately adjacent to the microgyrus generates epileptiform activates. The planned experiments will test potential mechanisms by which this hyperexcitability might occur, namely a decrease in GABAa receptor-mediated inhibition and enhanced excitatory synaptic interactions due to altered intracortical circuitry. Techniques will involve use of neocortical slices maintained in vitro, and application of patch-clamp methods for recording spontaneous and evoked whole cell currents from neurons visualized in 'thin' slices, and those in standard slice preparations. Neuroanatomic studies will be done on neurons filled with biocytin, and in experiments where orthograde and retrograde tracers will be used to examine connectivity in and around the microgyrus. The results promise to provide new information about mechanisms for development of epileptogenesis in a prototypic cortical malformation.

DESCRIPTION (provided by applicant): Inhibitory interneurons play a number of key roles in normal neocortical function. For example, they shape sensory receptive fields and drive high frequency gamma oscillations. On the other hand, defects in their function can lead to seizures. We have examined the properties of two major functional interneuronal subclasses: fast spiking (FS) and low-threshold spike firing (ITS) neurons of rat neocortical layer V. Data indicate that each group expresses a novel form of self inhibition, namely autaptic inhibitory responses in FS cells and an endocannabinoid-mediated slow self-inhibition in ITS interneurons. We will address two major questions relevant to self-inhibition of neocortical interneurons: 1) What are the roles of FS cell autapses in regulating precision of spike timing and in coordinating fast network synchrony? 2) What are the mechanisms leading to long-lasting inhibition in ITS neurons, and the physiological conditions necessary for its induction? Overall these two experimental aims will address the central themes of this grant, the mechanisms that modulate and control neocortical interneuronal activities and the functional consequences of such modulation on neocortical circuit function. Experimental approaches will include: single and paired whole cell voltage- and current-clamp recordings and perforated-patch recordings from visualized interneurons in rat neocortical slices; intracellular labeling with biocytin; intracellular and extracellular application of ions and pharmaceutical agents to affect transmitter release and receptor function; and use of dynamic clamp. Results will lead to a better understanding of synaptic modulation of two major subclasses of neocortical interneurons and provide information regarding GABAergic regulation of neocortical excitability relevant to both normal and pathophysiological cortical function.

DESCRIPTION (provided by applicant): Electrophysiological and anatomical studies will be done on a rat model of post-traumatic epilepsy to address questions of alterations in excitatory and inhibitory mechanisms within the neocortex that might lead to epilepsy following injury. Epileptogenic lesions are produced in adult rats by making partial cortical isolations that generate abnormal discharges within 2-3 weeks. Studies are planned to assess alterations in GABA-A receptor-mediated inhibition that might be produced by changes in the chloride gradient of post-synaptic neurons, due to a down regulation of the K/Cl co-transporter, KCC2. Other experiments will assess the excitatory and inhibitory synaptic inputs to inhibitory interneurons within the epileptogenic cortex, to test the hypothesis that these cells receive less excitatory and/or more inhibitory functional innervation. In other experiments, connectivity within layer V of the abnormal cortex will be studied to determine whether axonal sprouting results in abnormal functional excitatory connectivity. Techniques employed will include immunocytochemistry, whole cell recordings of currents and potentials in cortical neurons, minimal stimulation and the application of scanning laser photolysis of caged glutamate. Results are relevant to the mechanisms of epilepsy that develop following brain trauma. The long-term goal is to use this information to find strategies that will prevent post-traumatic epilepsy.

DESCRIPTION (provided by applicant): Traumatic brain injury (TBI) results in abnormalities in cerebral cortex and disorders of sensory and motor function, cognitive abnormalities and epilepsy. The potential development of epilepsy by the large number of individuals, who have survived severe concussive injury during recent conflicts, emphasizes the need for understanding the underlying pathophysiological processes and the development of prophylactic strategies (Garga and Lowenstein, 2006). A goal of this project is to obtain basic information on approaches that may improve or prevent such posttraumatic abnormalities. Injury to nerve cells that release the chemical transmitter GABA is a common result of TBI. Improvement in the structure and function of these inhibitory cells, interneurons, may prevent some of the consequences of injury, including epilepsy. Preliminary results show that fast-spiking (FS) inhibitory interneurons, the most common type of interneuron in cortex, have abnormal nerve processes and defects in releasing GABA in areas of cortical injury produced by partially cutting connections with surrounding brain (undercuts). Undercut cortex becomes hyperexcitable due to this and other defects and often generates epileptiform electrical activity that resembles EEG activity in human focal epilepsy. A neurotrophic protein BDNF, and its receptor TrkB, are important for development, growth and maintenance of interneurons, and are reduced in the injured area, leading to the hypothesis that TrkB activation may correct abnormalities in FS or other interneurons and improve function in the injured cortex. To test this hypothesis, undercuts will be made in anesthetized rodents, and animals treated with a newly- engineered small molecule, LM22A-4, that enters the brain and activates the TrkB receptor. After treatment for ~2 weeks, rodents are re-anesthetized, and standard in vitro slice and patch clamp techniques used to obtain recordings from single interneurons and excitatory cells in areas of injury from drug-treated animals and saline controls. Activities of individual nerve cells and large groups of neurons (field potentials) will be analyzed with appropriate software. Laser-activated release of the excitatory chemical transmitter, glutamate, will be used to map changes in excitatory and inhibitory connections in neural networks within individual slices, and effects of chronic LM22A-4 treatment. The structure of single cells will be measured after filling them with a marker called biocytin, staining slices with antibodies, and using computer-controlled microscopic imaging techniques, including light and confocal microscopy. The aims of these experiments are to determine whether activation of the TrkB receptor will improve the anatomical and functional abnormalities in FS interneurons, restore normal release of GABA and favorably affect nerve circuits in the injured cortex.

DESCRIPTION (provided by applicant): The effects of gabapentin and thrombospondins on enhanced excitatory connectivity, new synapse formation and epileptogenesis after neocortical injury sprouting of new excitatory wiring between neurons in cerebral cortex and hippocampus, changes in glial cells and formation of excitatory synapses are consequences of brain injury that are thought to contribute to epilepsy in animal models and human brain. There is no effective prophylactic treatment available to prevent epileptogenesis after brain injury. The planned experiments focus on a new approach for limiting the development of the increased excitatory connections and epilepsy after cortical trauma. Astrocyte-secreted thrombospondins (TSPs) are involved in new excitatory synapse formation during development and after cortical injury. Experiments will test the hypothesis that pregabalin (PGB) (Lyrica), an approved drug that interferes with the binding of TSPs to their alpha2delta-1 receptor, will decrease excitatory synapse formation and sprouting and limit development of epileptiform activity. The effects of PGB and TSPs will be explored in the partial cortical isolation (undercut, UC) model of epileptogenic neocortical injury in naïve mice, in TSP knockout mice and alpha2delta-1 overexpressing epileptic mice. The incidence of epileptiform activity recorded in in vitro cortical slices after injury, sprouting of axons, density of excitatory synapses and network connectivity will be measured using electrophysiological and anatomical techniques. Laser scanning photostimulation of caged glutamate will be used in conjunction with whole cell recordings to map excitatory connections in cortical slices. A possible link between excessive neuronal activity and increases in TSPs, the alpha2delta-1 receptor and new synapse formation will be studied in naïve or injured cortex. The effects of PGB treatment after cortical injury in vivo on these measures will be assessed to test the hypothesis that the drug will decrease the structural and functional abnormalities that follow brain trauma and lead to the development of epilepsy. Relevance: Posttraumatic epilepsy is a prominent consequence of serious neocortical or hippocampal injury that alters neuronal and glial structure and function and induces hyperexcitability in cortical circuits. Unfortunately, treatment is often ineffective at relieving seizures once they occur and no drug is available that will prevent the epileptogenic processes that lead to posttraumatic epilepsy. Results of these experiments will contribute to understanding cellular and circuit effects of cortical injury, and provide new information about a potential role for gabapentin and pregabalin to prevent development of epilepsy after brain injury.

Abnormalities in parvalbumin (PV) and somatostatin (SOM) interneurons are reported in a number of neurological disorders, including epilepsy. Therapy that improves function of defective interneurons is not available. Structural development and maintenance of interneurons is dependent on trophic support provided by brain derived neurotrophic factor (BDNF) activation of TrkB receptors. In the undercut (UC) model of epileptogenic neocortical injury, chronic activation of TrkB-Rs with a selective small molecule partial agonist (LM22A-4, ?LM? below) has long-term effects to reverse structural and functional abnormalities in inhibitory terminals of PV interneurons, enhance GABA release and increase the threshold for evoking epileptiform activity and seizures. In order to determine whether these effects will be applicable to treatment or prevention of epilepsy in other models with different causes for seizures, such as genetic epilepsies, the Dravet syndrome (DS) mouse will be used in some experiments. Decreases in a membrane sodium channel in PV interneurons in DS mice causes decreased release of the inhibitory transmitter GABA, and development of spontaneous and high temperature-induced seizures. A variety of experimental approaches in DS and UC mice will be used to determine whether chronic treatment with LM, by increasing GABA release from nerve terminals of SOM and PV interneurons, or inducing new inhibitory synapse formation, will enhance inhibition in cortical networks and suppress epileptiform discharges: 1) immunocytochemistry and confocal imaging will be used to assess alterations in PV and SOM presynaptic terminals, including changes in expression of VGAT- and GAD65/67- IR, and the calcium sensor protein synaptotagmin 2; 2) analysis of density of SOM/- and PV/gephyrin close appositions to test for new inhibitory synapse formation induced by TrkB activation; 3) electrophysiological analysis of basic properties of inhibitory synaptic transmission from PV interneurons to pyramidal neurons of in vitro slices to detect effects of TrkB activation on unitary IPSCs, release probability and transmission failures; 3) laser scanning photostimulation of cortical slices from PV/CHR2 and SOM/CHR2 mice to map the distribution and strength of inhibitory connectivity in neocortical inhibitory circuits; and 4) video/EEG monitoring of implanted mice to assess effects of treatment with LM on spontaneous seizures and hyperthermia-induced seizures. Results of these experiments will provide information about mechanisms leading from interneuronal abnormalities to development of epilepsy and a potential approach to prophylaxis of epileptogenesis by enhancing trophic support of interneurons.