Abstracts

Status epilepticus (SE) is a progressive condition in which a reduction in GABA-mediated inhibition facilitates the self-sustaining nature of the seizure. An activity-dependent increase in the rate of internalization of postsynaptic GABA[sub]A[/sub] receptors (GABARs) is an attractive mechanism to explain the reduction. However, it is not known whether the rate of GABAR internalization is activity-dependent. Electrophysiological and immunocytochemical techniques were used to examine the effect of SE on GABAergic synaptic transmission and the internalization of GABARs in a network of cultured hippocampal neurons. Hippocampal pyramidal neurons were cultured per the methods of Goslin and Banker. Removing MgCl[sub]2[/sub] from the extracellular media results in sustained epileptiform bursting ([italic]in vitro[/italic] SE). [underline]Electrophysiology:[/underline] Miniature inhibitory postsynaptic currents (mIPSCs) and whole-cell GABA currents were recorded using standard whole-cell patch clamp techniques. [underline]Internalization assay:[/underline] The GABARs on living cultured neurons were tagged with an antibody directed against the GABAR [beta]2/3 subunit. After tagging, the neurons were incubated in an antibody-free external media at 37C allowing antibody-tagged receptors to undergo endocytosis. Following fixation, the neurons were exposed to secondary antibodies before and after permeabilization permitting antibody-tagged surface and internalized receptors to be independently identified. The surface and internalized immunoreactivity was measured and used to determine the percentage of internalized tagged-receptors. [underline]Synaptic transmission:[/underline] Compared to controls, [italic]in vitro[/italic] SE of [gt]2 hours resulted in a 30% reduction in mIPSC amplitude (53.8 [plusmn] 1.6 vs. 36.2 [plusmn] 1.5 pA) and an 80% reduction in the maximal whole-cell GABA current (3897 [plusmn] 364 vs. 685 [plusmn] 48 pA). [underline]Rate of internalization:[/underline] The rate of GABAR internalization was assessed under the following conditions: (1) control external media, (2) neuronal activity inhibited with TTX, and (3) [italic]in vitro[/italic] SE. Under all conditions, surface immunoreactivity diminished and internalized immunoreactivity increased with time. At 30 minutes, in control external media, approximately 50% of the receptors were internalized. This fraction remained stable over the next 30 minutes. When neuronal activity was inhibited, the percentage of internalized receptors at all time points (10, 20, 30, and 60 minutes) was reduced reaching a plateau of 35%. In contrast, during [italic]in vitro[/italic] SE, the percentage of internalized receptors was increased at all time points, with a plateau of 60% internalization. A reduction in GABA-mediated inhibition occurs during [italic]in vitro[/italic] SE. A concurrent, activity-dependent increase in the rate of GABAR internalization likely contributes to this reduction in GABA-mediated inhibition. Further studies are required to better define the mechanisms involved in the activity-dependent internalization of GABARs. (Supported by NINDS.)