Friday, May 9, 2025
5/9/2025
Na+/Ca2+ exchanger remodeling in alcohol withdrawal seizures - Generalized tonic-clonic seizures (GTCSs), the most prevalent type of alcohol withdrawal‒induced seizures (AWSs), are commonly resistant to current anticonvulsants, nevertheless, their underlying mechanisms are poorly understood. Our long-term goal is to understand how altered Ca2+ signaling contributes to the neuronal hyperexcitability that causes increased AWSs susceptibility. We have previously reported upregulation of proteins associated to L-type (CaV1.3) and P-type (CaV2.1) voltage-gated Ca2+ (CaV) channels in inferior colliculus (IC) neurons when AWSs prevalence peaks but not before the onset of AWSs susceptibility. The upregulation of CaV channels leading to increased intracellular Ca2+ ([Ca2+]i) and altered Ca2+ homeostasis suggests that the Na+/Ca2+ exchanger, a bidirectional membrane ion transporter that regulates [Ca2+]i would preferentially operate in the forward mode (NCXfwd) to extrude Ca2+ and restore Ca2+ homeostasis. Surprisingly, NCX operates in the reverse mode (NCXrev) causing Ca2+ influx into IC neurons prior to the onset of AWSs susceptibility and when AWSs prevalence peaks. Thus, changes in NCX activity possibly play a critical role in AWS initiation. The experiments proposed here will determine the precise contribution of NCX type1 (NCX1) to neuronal hyperexcitability and AWSs susceptibility, providing specific target for developing novel therapeutics. Our working hypothesis is that Ca2+ influx in IC neurons via NCXrev is essential for generating the epileptiform bursts that initiate an AWS. To test this hypothesis—and building on our preliminary data and prior publications— we will combine in vivo pharmacology with molecular genetics, electrophysiology, molecular biology, and behavioral analysis in three specific aims. First, we will determine the extent to which NCXrev contributes to generating the epileptiform bursts in IC neurons following alcohol withdrawal; in addition, we will investigate how intracellular Na+ concentration as well as L- and P-type CaV channels contribute to increased NCXrev activity during the course of alcohol withdrawal. Second, we will determine the extent to which changes in NCX1 gene expression and cell surface protein levels in IC neurons are associated with increased seizure susceptibility during the course of alcohol withdrawal. Third, we will evaluate AWSs generated in rats in which NCX1 expression is deleted selectively in IC neurons. Furthermore, we will measure AWSs in rats in which hyperexcitability mediated by glutamatergic IC neurons is suppressed using optogenetics. Our findings will provide key insight into NCX1 role on seizure activity, the mechanisms that initiate AWSs, and the mechanisms that underlie AWSs and other types of GTCSs.
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