Alcohol withdrawal (WD) produces a range of dangerous clinical symptoms, including intense seizures. Hyperexcitability underlying seizures is produced by an array of intrinsic membrane properties that are disrupted by ethanol (EtOH). Prior work has demonstrated that chronic EtOH exposure and WD produce an up-regulation of ion channel proteins and a gain of function that promotes WD seizure. A remaining gap in our understanding of WD-related seizure is a testable model that places cellular changes in a network context. WD produces upregulation and increased bursting in midline thalamic nuclei. In hippocampus, mammalian target of rapamycin Complex 1 (mTORC1) is activated in CA1 neurons during WD, represses translation of Kv1.1, and results in reduced inhibition that we hypothesize will allow invasion of thalamic bursts and increased epileptiform population discharges. We have developed a new model of network excitability that will allow us to study the emergence, time course and molecular underpinnings of EtOH WD hyperexcitability and seizure. We will address the following aims:
In Aim 1, we will determine the intrinsic properties contributing to membrane hyperexcitability in midline thalamus and CA1 due to ethanol WD seizure. Using voltage clamp recordings in an in vitro preparation coupled with pharmacological approaches, we will determine whether epileptiform discharges in WD are ultimately dependent on a progressive imbalance between excitatory burst discharges in thalamus (which depend on PKC), and reduced K+ currents in CA1 pyramidal cells (which are controlled by mTOR).
In Aim 2, we will Determine important regulators of dendritic excitability in thalamus and CA1 in EtOH WD seizure. mTOR signaling is implicated in the development of spontaneous seizures in epilepsy, and we show data that it is active during WD. Using molecular approaches, we will toggle mTOR activity in the presence and absence of protein synthesis inhibitors. We will test whether mTORC represses translation of Kv1.1, as suggested by our preliminary data.
In Aim 3, we bring together the cellular and molecular findings to determine the effects of WD-mediated changes to network excitability and seizure susceptibility in vivo. Using a novel optogenetic approach, we will test whether stimulation of the thalamo-HC pathway during WD will elicit enhanced epileptiform activity compared to controls that will depend on patterned activity at facilitated CA1 synapses. We expect that disruptions of mTORC1 will modify or reverse WD-mediated excitability. Seizure threshold is significantly reduced during repeated EtOH WD and we will use this fact to test the hypothesis that drugs effective against WD-induced hyper-excitability will also be effective at raising seizure thresholds to baseline levels. Success in these experiments will provide a more comprehensive understanding of how brief spindle episodes and spike wave complexes promote or support tonic-clonic WD seizures ? which could lead to the identification of novel pathways and associated drug targets that will provide a means to prevent WD seizure, and to more effectively treat it.
The proposed research is relevant to public health because the study of the basic mechanisms of seizure has broad applications to neurological disease, including numerous epilepsy types, substance abuse and withdrawal syndromes. The proposal has high translational value, and is relevant to the NIH mission to gain knowledge about the nature and behavior of living systems and to apply that knowledge to improve diagnosis, prevention, and treatment of alcohol-related problems, including alcohol use disorder, across the lifespan.
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