Nicholas P. Poolos - US grants

Epilepsy is a disease of neuronal hyperexcitability. Several models of epilepsy at the cellular level have pointed to the CA3 hippocampal pyramidal cell as a locus of epileptogenic burst firing. Recent recordings in CA1 hippocampal and neocortical pyramidal cells have shown that the dendrites of these cells are capable of firing action potentials independently of the cell soma, and that somatic action potentials are actively propagated into the dendrites where they can influence dendritic excitability. This study proposes to examine active electrogenesis in the dendrites of CA3 pyramidal cells in order to determine whether these dendrites possess active properties similar to those of CA1 pyramidal cells, and how these properties might contribute to epileptiform firing. Action potential propagation will be visualized in CA3 cells using dendritic patch clamping and imaging of intracellular calcium concentration with fluorescent dyes. These techniques should allow the localization of the initiation sites of both sodium- and calcium-dependent action potentials within the dendrites. The firing and invasion of the dendrites by somatic action potentials will also be assessed under both normal and epileptogenic conditions induced by potassium channel blockers and GABAergic disinhibition. The influence of these "backpropagating" action potentials on dendritic excitability will be studied, and the distribution and pharmacology of dendritic calcium channels will be mapped. It is hoped that these studies will provide further evidence for the role that active dendrites play in contributing to the overall behavior of the single neuron, particularly under conditions which lead to epileptiform firing.

DESCRIPTION (provided by applicant): Epilepsy is a disease of abnormal neuronal excitability, but the causes of this hyperexcitability remain largely unknown. Our understanding of the intrinsic determinants of neuronal excitability has significantly improved in recent years due to technical advances which allow electrophysiological study of neuronal dendrites, such as those of CA1 hippocampal and neocortical pyramidal neurons. This proposal investigates the possibility that temporal lobe epilepsy is associated with altered biophysical properties of a voltage-gated channel, which is primarily localized to dendrites, the h-channel or Ih. Prior studies have found that Ih in hippocampal pyramidal neurons can be altered by a single prolonged seizure, and that Ih is a target of anticonvulsant action. We propose studying Ih in hippocampal pyramidal neuron dendrites to determine if its properties are altered in an animal model of chronic epilepsy. Because prior work by the PI and others has shown that dendritic Ih reduces overall pyramidal neuron excitability, our central hypothesis will be that Ih may be down-regulated in the dendrites of pyramidal neurons in epileptic animals, producing neuronal hyperexcitability. The studies proposed involve whole-cell and cell-attached patch clamp electrophysiology in the soma and dendrites of CA1 hippocampal pyramidal neurons prepared using brain slice techniques. Specifically, we will answer the following questions: 1) How is Ih modulated under normal conditions in pyramidal neuron dendrites? 2) Are Ih properties altered in pyramidal neuron dendrites from epileptic animals? 3) Is dendritic Ih differentially modulated in chronic epilepsy? These studies may provide further evidence for the hypothesis that epilepsy results in part from changes in the intrinsic excitability of neurons, and may suggest novel targets, such as the h-channel or its modulators, in the treatment of epilepsy. The PI has recently started a laboratory at the University of Washington devoted to cellular neurophysiology, and treats adults with epilepsy as part of the Regional Epilepsy Center there. If funded, this proposal will enable the PI's transition to an independent clinician-scientist with a career focus on both basic and clinical aspects of epilepsy.

DESCRIPTION (provided by applicant): Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are voltage-gated ion channels that modulate excitability in several brain regions involved in the pathogenesis of epilepsy, including hippocampus, neocortex, and thalamus. The preponderance of evidence shows that downregulation of Ih, the current generated by HCN channels, is associated with neuronal hyperexcitability and epilepsy. In the prior funding period of this project, the onset of epilepsy in an animal model was found to be associated with loss of HCN channel expression, and downregulation of HCN channel gating (i.e. hyperpolarization of Ih voltage-dependent activation). This latter change in HCN channel properties may be particularly important in the generation of seizures, as lamotrigine, an antiepileptic drug, produces upregulation of HCN channel gating as part of its antiepileptic action. A novel modulator of HCN channel gating, p38 mitogen- activated kinase (p38 MAPK), was characterized as well, with inhibition of p38 MAPK causing downregulation of HCN gating. In the present proposal, the links between phosphorylation (i.e. kinase or phosphatase) signaling and HCN channels will be explored. The overall hypothesis is that the downregulation of HCN channel gating that occurs in epilepsy may be due to loss of kinase activity, such as that of p38 MAPK, or to increased phosphatase activity. We will also explore whether direct modulation of p38 MAPK activity exerts an antiepileptic action in vivo. Specifically, we will answer the following questions: 1) Is altered HCN channel gating in epilepsy associated with loss of kinase activity? 2) Is altered HCN channel gating in epilepsy associated with increased phosphatase activity? 3) Is upregulation of HCN gating via activation of p38 MAPK signaling effective in an animal model of temporal lobe epilepsy (TLE)? To answer these questions, we will use cellular electrophysiology techniques in the brain slice preparation, biochemical techniques to assay kinase and phosphatase activity, and long-term video-electroencephalography (VEEG) in the pilocarpine animal model of epilepsy. The outcome of these experiments should increase our understanding of the molecular mechanisms by which seizures alter ion channel biophysical properties, determine whether epilepsy is associated with derangement of phosphorylation signaling pathways, and explore possible novel antiepileptic treatment strategies. PUBLIC HEALTH RELEVANCE: The current proposal will study the mechanisms of epilepsy at cellular and molecular levels. Epilepsy is one of the most common neurological diseases, affecting nearly 1% of the population, and causing significant disability in the 30% of epilepsy patients whose seizures are uncontrolled by existing medication. The outcome of this study may identify specific biochemical pathways that could be targeted for development of novel antiepileptic drugs, improving the likelihood that currently poorly-controlled patients may one day be seizure-free.

DESCRIPTION (provided by applicant): While numerous antiepileptic drugs have a high likelihood of producing seizure freedom in new-onset epilepsy, refractory epilepsy by definition represents that 1/3rd of all epilepsy patients who cannot be successfully treated by AEDs, either alone or in combination, a situation that has persisted over decades despite the introduction of many new AEDs. However it has been widely hypothesized that some AEDs might be more effective used in combination, such that their action together exceeds the predicted sum of their individual effects, otherwise known as synergism. We recently published a large-scale retrospective human study that demonstrated that only a single combination of AEDs, lamotrigine (LTG) plus valproate (VPA), was significantly more effective in treating refractory epilepsy, potentially exhibiting synergism, while LTG in combination with carbamazepine (CBZ) appeared to be less effective than the sum of their parts, thus exhibiting antagonism. In this study, we wish to translate these clinical findings to an animal model of refractory epilepsy in order to understand the cellular mechanisms. We will first rigorously determine whether the efficacy of LTG/VPA represents true pharmacodynamic synergism, or results from a pharmacokinetic interaction. We will then seek evidence for a novel hypothesis as to the failure of most AED regimens in refractory epilepsy: that many AEDs constrain the excitability of inhibitory interneurons as well as excitatory principal neurons, and that this off-target action promotes AED refractoriness. The potential outcome of this study will be the translation from bedside- to-bench of a novel clinical finding-understanding the mechanisms of which could lead to an improved approach to AED design.

Project summary About 1/3rd of people with epilepsy are medically refractory, that is, do not achieve seizure freedom with antiepileptic drugs (AEDs). There are few evidence-based guidelines on the optimal treatment of refractory patients. In previous work, we studied an institutionalized, developmentally disabled patient population in Washington State and discovered that of the most frequently-used AED regimens, only the combination of lamotrigine (LTG) and valproate (VPA) demonstrated significant benefit in refractory epilepsy. A caveat to this finding was the specialized patient population in which the study was conducted. In this proposal, we will use two complementary approaches in community-based patients to search for AED regimens with superior efficacy in refractory patients, so to determine whether our previous findings are applicable to the general refractory epilepsy population. In the first Specific Aim, we will study individuals with epilepsy who use either of two online seizure diaries to track their seizure occurrences and AED usage. In the second Aim, we also study the entire outpatient epilepsy population of the University of Washington Regional Epilepsy Center, using automated analysis of the electronic health record, to determine which AED regimens had the highest success rate in producing seizure freedom. The potential outcome of this study will be the identification of AED regimens that are the most effective in a prevalent but difficult to treat epilepsy population.