Abstract
Altered GABAergic inhibitory tone has been observed in association with a number of both acute and chronic models of epilepsy and is believed to be the result, in part, of a decrease in function of the postsynaptic GABAA receptor (GABAAR). This study was carried out to investigate if alterations in receptor internalization contribute to the decrease in GABAAR function observed with epilepsy, utilizing the hippocampal neuronal culture model of low-Mg2+-induced spontaneous recurrent epileptiform discharges (SREDs). Analysis of GABAAR function in “epileptic” cultures showed a 62% reduction in [3H]flunitrazepam binding to the GABAA α receptor subunit and a 50% decrease in GABA currents when compared with controls. Confocal microscopy analysis of immunohistochemical staining of GABAAR β2/β3 subunit expression revealed approximately a 30% decrease of membrane staining in hippocampal cultures displaying SREDs immediately after low-Mg2+ treatment and in the chronic epileptic state. Low-Mg2+-treated cultures internalized antibody labeled GABAA receptor with an increase in rate of 68% from control. Inhibition of GABAAR endocytosis in epileptic cultures resulted in both a recovery to control levels of membrane GABAA β2/β3 immunostaining and a total blockade of SREDs. These results indicate that altered GABAAR endocytosis contributes to the decrease in GABAAR expression and function observed in this in vitro model of epilepsy and plays a role in causing and maintaining SREDs. Understanding the mechanisms underlying altered GABAA R recycling may offer new insights into the pathophysiology of epilepsy and provide novel therapeutic strategies to treat this major neurological condition.
Footnotes
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This work was supported by a National Institute of Neurological Disorders and Stroke grant (RO1-NS23350 to R.J.D.), by the Epilepsy Program Project award (P50-NS25630 to R.J.D), by the Milton L. Markel Alzheimer's Disease Research Fund, and by the Sophie and Nathan Gumenick Neuroscience Research Fund.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.104.068478.
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ABBREVIATIONS: AE, acquired epilepsy; SE, status epilepticus; GABAAR, γ-aminobutyric acid type A receptors; SRED, spontaneous recurrent epileptiform discharge; BRS, basal recording solution; BZ, benzodiazepine; FNZ, flunitrazepam; kDa, kilodalton(s); PRD, proline-rich domain; GDPβS, guanosine 5′-[β-thio] diphosphate; DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate.
- Received March 16, 2004.
- Accepted April 14, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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