Trafficking of GABA(A) receptors, loss of inhibition, and a mechanism for pharmacoresistance in status epilepticus

Abstract

During status epilepticus (SE), GABAergic mechanisms fail and seizures become self-sustaining and pharmacoresistant. During lithiumpilocarpine-induced SE, our studies of postsynaptic GABA(A) receptors in dentate gyrus granule cells show a reduction in the amplitude of miniature IPSCs (mIPSCs). Anatomical studies show a reduction in the colocalization of the beta2/beta3 and gamma2 subunits of GABA(A) receptors with the presynaptic marker synaptophysin and an increase in the proportion of those subunits in the interior of dentate granule cells and other hippocampal neurons with SE. Unlike synaptic mIPSCs, the amplitude of extrasynaptic GABA(A) tonic currents is augmented during SE. Mathematical modeling suggests that the change of mIPSCs with SE reflects a decrease in the number of functional postsynaptic GABA(A) receptors. It also suggests that increases in extracellular [GABA] during SE can account for the tonic current changes and can affect postsynaptic receptor kinetics with a loss of paired-pulse inhibition. GABA exposure mimics the effects of SE on mIPSC and tonic GABA(A) current amplitudes in granule cells, consistent with the model predictions. These results provide a potential mechanism for the inhibitory loss that characterizes initiation of SE and for the pharmacoresistance to benzodiazepines, as a reduction of available functional GABA(A) postsynaptic receptors. Novel therapies for SE might be directed toward prevention or reversal of these losses.

Figure 1.

Subcellular distribution of GABA_A β2/β3 subunits with SE. Hippocampal sections of control (left) and SE (right) stained with an antibody against GABA_A β2/β3 subunits (red, top) and an antibody against the presynaptic marker synaptophysin (green, middle). Overlaps between presynaptic synaptophysin and postsynaptic GABA_A subunit appear yellow (bottom). Note that fewer GABA_A subunit puncta colocalize with synaptophysin with greater internalization of the red receptor subunits relative to the green synaptophysin outline during SE (right, bottom), suggesting trafficking of GABA_A subunits. sg, Stratum granulosum; h, hilus.

Figure 2.

Distribution of GABA_A γ2 subunits for SE. A, Hippocampal sections of control (left) and SE (right) stained with an antibody against GABA_A γ2 subunits (red, top) and an antibody against the presynaptic marker synaptophysin (green, middle). Colocalizations in yellow represent overlaps between presynaptic synaptophysin and postsynaptic GABA_A subunit (bottom). B, Magnification of the bottom panel in A showing fewer yellow GABA_A subunit puncta colocalizations with synaptophysin and internalization of the red receptor subunits relative to the green synaptophysin outline during SE (right). sg, Stratum granulosum; h, hilus. Scale bars, 10 μm.

Figure 3.

mIPSCs from dentate gyrus granule cells of SE and controls. A, mIPSC mean traces from a typical granule cell from a control (solid line) and an SE animal (dotted line) demonstrating smaller amplitude and prolonged decay in the latter. B, Optimized computer model fits (dashed line) to mean and SD traces of mIPSCs (solid) from a typical control and an SE cell (see Table 1 for model values). C, Histogram for peak amplitudes of individual mIPSC events recorded from a control (left) and an SE (right) granule cell with the superimposed predicted distribution (filled circles) from model fits of B.

Figure 4.

Graphic representation of mIPSC model numerical values (Table 1). The model predictions for control (solid line) and SE (dashed line) amplitudes and time courses are shown for GABA release into the synaptic cleft (A), mean mIPSCs (B), and percentage of postsynaptic GABA_A receptors in desensitized states (C). Note the predicted prolongation of GABA exposure for SE in A and the significant persistence of receptors in desensitized states, with greater desensitization for SE in C.

Figure 5.

GABA_A tonic currents of granule cells after SE. A, Recordings from typical cells from control and SE animals. Note the increase mean (and baseline SD) of the tonic current for SE cells compared with controls, as revealed by the greater baseline shift with the addition of the GABA_A receptor antagonist SR95531. Tonic currents for both control and SE cells were accentuated by the addition of the GABA uptake inhibitor N0711 (10 μm) and GABA (1 μm). B, Optimized model fit for tonic current means and SDs from control (asterisk; n = 14 cells) and SE (cross; n = 12 cells) animals. The solid line curve was the optimization after adjusting N and i of Equation 3 to fit tonic current mean and SD results for control and SE cells. The circles represent model-predicted dose responses of tonic current mean and SD values (each circle correlates to a 1 μm increase in extracellular [GABA] to a total of 20 μm). To calibrate the dose-response predictions of the model, measured tonic current mean and SDs obtained from control experiments with perfusates of known [GABA] are superimposed (3, 5, and 10 μm with 10 μm NO711; boxes with error bars as ±SEM). C, Model-predicted GABA_A receptor peak open dose-response curves (left) and responses for step exposure to GABA (right) using receptor parameter values from optimized fits to mIPSCs of control cells, to mIPSCs of SE cells, and to tonic currents (see Table 1).

Figure 6.

Perforant path-evoked IPSCs and model-predicted paired-pulse responses for dentate granule cells. A, Typical evoked IPSC recording (solid line) in a control animal with optimized fit by a model (dashed line) for evoked activation (see Materials and Methods, Computer modeling). B, Model-predicted evoked paired-pulse responses (interstimulus interval of 40 ms) in granule cells using synaptic parameters from fits of mIPSCs from control (left) and SE (right) cells, also using elevated tonic extracellular [GABA] for SE conditions (estimated from increased tonic currents with SE). Predicted evoked currents (short-dashed line) and percentage of GABA_A receptor desensitization (long-dashed line) show loss of paired-pulse inhibition and greater desensitization under SE conditions (P2/P1 from 1.21 for control to 0.79 for SE parameters). For SE conditions in the model, the slight desensitization of synaptic receptors before the paired pulses is a result of 5 μm extracellular GABA.