Vijayalakshmi Santhakumar, MD, PhD - US grants

DESCRIPTION (provided by applicant): An estimated 200,000 new cases of epilepsy are diagnosed each year in the United States. Temporal lobe epilepsy, the most common epileptic syndrome, often develops following early unprovoked seizures and is particularly resistant to mainstream antiepileptic drugs. The hippocampal dentate gyrus is at the heart of the characteristic structural and functional changes that underlie temporal lobe epilepsy. A network of perisomatically projecting GABAergic interneurons regulates the excitability of dentate projection neurons, the granule cells. Activity and synchrony of inhibitory networks are governed by a combination of gap junctional and GABAergic chemical connections. However, whether GABAergic inhibition and electrical coupling among the perisomatic interneurons are modified during development of epilepsy and underlie the instability in network activity in epilepsy is yet to be examined. Additionally, dynamic changes in inhibitory and electrical coupling among interneurons are likely to determine the duration and spread of seizures. Understanding how activity patterns in the perisomatic inhibitory network are altered following status epilepticus and dynamically regulated during seizures will help evaluate whether pharmacological manipulation of gap junctions and GABA receptors would be effective in treating epilepsy. The hypothesis of this proposal is that status epilepticus (SE) alters non-synaptic and synaptic coupling between fast-spiking perisomatic dentate interneurons resulting in enhanced mutual inhibition which compromises feedback inhibition of projection neurons. It is further proposed that modulation of inhibitory currents and electrical coupling by pH changes that accompany neuronal activity undermine perisomatic inhibition during neuronal activity enhancing dentate excitability and contributing to epileptogenesis. The study will use pilocarpine induced status epilepticus to model development of acquired epilepsy, and a combination of anatomical, physiological and computational approaches to address the following specific questions. Aim 1 will identify the presence of tonic GABA currents in fast-spiking basket cells and examine whether post-status enhancement of tonic GABA currents compromise perisomatic inhibition of granule cells. Aim 2 will identify post-status changes in synaptic and electrical coupling among basket cells and their effects on dentate network excitability and synchrony. Aim 3 will test whether activity-dependent modulation of basket cell synaptic and non-synaptic coupling by acidic pH shifts accompanying neuronal activity undermines inhibition and contributes to epileptogenesis after status epilepticus. It is anticipated that the study will identify fundamental mechanisms underlying dynamical instability of dentate network activity in acquired epilepsy. PUBLIC HEALTH RELEVANCE: Acquired temporal lobe epilepsy is a disorder affecting over 1.5 million patients and is associated with long term decrease in quality of life. The experiments proposed in this study will determine if dynamic decreases in inhibition during seizures contributes to prolonged neuronal activity and development of epilepsy. It is expected that understanding the changes in inhibitory circuit function in epilepsy will lead to novel alternatives to manage patients with early unprovoked seizures and reduce the risk for developing epilepsy.

Project Summary: Neurological disorders such as epilepsy and memory loss that develop several years after traumatic brain injury are a major source of physical disability and economic burden after brain trauma. The time window between the initial insult and the disease suggest that progressive changes that occur after brain injury underlie neurological disease and that early interventions might prevent these debilitating outcomes. The hippocampal dentate gyrus is the major focus of neuronal damage and increased excitability after concussive brain injury and in post-traumatic temporal lobe epilepsy. Apart from injuring neurons, traumatic release of endogenous molecules from disrupted cells and extracellular matrix can activate pattern-recognition receptors of the innate immune system including Toll-like receptors. Certain TLR subtypes, including TLR4 are expressed in neurons and regulate neurogenesis and cell death. The central hypothesis of this proposal is that, early post-injury increase in activation of neuronal TLR4 alters excitability and leads to excitotoxic damage of specific dentate neuronal types and facilitating acute and chronic increases in network excitability. Using the rodent fluid percussion injury model of concussive brain trauma and current physiological techniques, Aim 1 will distinguish the cellular, signaling and channel mechanisms underlying TLR4 modulation of neuronal excitability in the normal brain and early after brain injury. Aim 2 will determine whether TLR4 activation in specific interneuronal populations contributes to excitotoxic injury and loss of certain interneuronal subtypes. Finally, Aim 3 will use a combination of histological, physiological and behavioral assays to test whether selective TLR4 antagonists reduce long-term susceptibility to epilepsy and memory deficits after brain injury. It is anticipated that the proposed studies will identify novel roles for perturbed TLR4 signaling in post-traumatic pathology and generate strategies for targeted treatment to improve the long-term neurological outcome after traumatic brain injury while preserving normal physiology. Such preventive strategies will greatly improve the quality of life of patients after brain injury and, in keeping with the NINDS mission, decrease the burden that post-traumatic neurological diseases place on the health care system.

Project Summary: Temporal lobe epilepsy (TLE) develops in a third of over 300,000 patients with a first seizure and over 30% of cases are resistant to drugs contributing to a significant disability. Presence of a therapeutic time window between the initial insult and development of epilepsy suggests that improved mechanistic understanding of early pathological process may enable prevention of epileptogenesis and associated co-morbidities. While sclerosis of the hippocampal dentate gyrus characterizes late stage TLE, cell loss, network reorganization and deficient inhibition in the dentate gyrus occur soon after insults that progress to TLE. In particular, the dentate inhibitory gate which limits GC activity throughput is compromised early in acquired TLE. However, what cells and circuits make up the dentate inhibitory gate and how this is compromised after seizures is not fully understood. Recently, a new class of neurons, semilunar granule cells (SGCs) were proposed as drivers of sustained dentate feedback inhibition. Although SGC-like neuros are observed in multiple species including humans and are activated during behaviors, the development, molecular identity, and connectivity of SGCs are not known making it difficult to determine their role in dentate function and disease. The limited literature and our pilot data that SGCs input and output connections are distinct from granule cells indicating that they play a unique role in dentate processing. This study will test the hypothesis that SGCs from a parallel dentate circuit that strengthens inhibition in the normal brain. We further propose that cellular and network changes after seizures compromise SGC mediated inhibition and augment their excitatory effects contributing to epilepsy and memory deficits. Combining morphometry, Patch-seq transcriptomics, electro- and optophysiology in transgenic mouse lines subject to experimental epilepsy and computational modeling will allow us to test the above hypothesis. Aim 1 will define the cellular and circuit identity of SGCs and determine molecular markers. Aim 2 will determine if the SGC excitatory circuit is strengthened and feedback inhibitory circuit compromised after status epilepticus. Finally, Aim 3 will examine the normal and seizure-induced development of SGCs and their contribution to dentate memory processing. On completion the studies will eliminate specific knowledge gaps in how the dentate circuit functions in behaviors and epilepsy, in keeping with the NINDS mission, and provide information needed to prevent collapse of dentate inhibition soon after seizures and prevent development of epilepsy and memory co-morbidities.