Post-Hypoxic Regulation of GABA-A Receptor Function

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Stroke and hypoxia frequently cause seizures or myoclonus, disorders of excessive neuronal excitability. Hypoxia-induced hyperexcitability is linked to dysfunction of inhibitory GABAA receptors (GABAARs), but the underlying mechanisms are poorly understood. GABAARs are chloride channels activated by y-aminobutyric acid (GABA), composed of subunits that determine their pharmacology and kinetic properties. We have studied the effects of hypoxia on GABAAR function and subunit mRNA expression in NT2-N neuronal cells and primary cortical neurons in vitro. Maximal GABA-evoked currents increased 1 h after transient hypoxia but then decreased to 60% of control after 48 h, associated with reductions in a1, a5, 02 and y2 subunit mRNAs. These changes are associated with induction of the bHLH transcription factor, hypoxia-inducible factor-1a(HIF1a) and calcium entry via voltage-gated channels opened by hypoxia-induced depolarizaton. Our long-term goal is to understand the mechanisms of hypoxia-induced GABAAR neuroplasticity, with these specific aims: 1. Determine the mechanisms underlying the early increase in GABAAR current after hypoxia. Hypothesis 1 is that the initial increase in GABAAR current is related to increased GABAAR channel density or phosphorylation. We will use whole-cell, perforated patch and single channel recordings to measure GABAAR currents, pharmacology, and single channel properties of post-hypoxic GABAAR currents. 2. Determine whether transcriptional changes account for the late reduction in GABAAR currents. Hypothesis 2 is that the reduction in GABAAR currents 48 h after hypoxia is related to altered GABAAR subunit transcription, and that specific subunit changes account for altered GABAAR pharmacology. We will use whole-cell recordings and RT-PCR to assess changes in GABAAR pharmacology and subunit expression. 3. Determine whether HIF-1a is involved in altered GABAAR subunit expression. Hypothesis 3 is that hypoxic induction of HIF-1a participates in regulating GABAAR subunit expression in concert with other signaling mechanisms. We will determine a. whether hypoxia alters HIF-1a expression, b. whether elevated HIF-1a reproduces hypoxia-related GABAR changes, and c. whether voltage-gated calcium channels mediate GABAAR regulation. These studies implicate a novel mechanism underlying post- hypoxic hyperexcitability, and could result in new treatments for post-hypoxic seizures and myoclonus.