Abstracts

Rationale: Seizure-evolution often progresses from fast-rhythmic to slow periodic synchronization before postictal termination.  Because GABAergic changes are rapid, involve seizure-initiation, and can persist, the role of functional changes in GABA-ARs and dynamic shifts in patterns of activation at synaptic and extrasynaptic sites is explored as a basis for circuit transitions during single seizures and with progression to steady-state status epilepticus (SE). Methods: Miniature, spontaneous, and evoked IPSCs as well as GABA-AR tonic currents were recorded from dentate gyrus granule cells in hippocampal slices using visualized whole-cell patch-clamp.  Recordings were performed in vitro before and after 2 Hz continuous / 20 Hz for 10 sec/min perforant path stimulation (PPS) and/or GABA uptake blockade or GABA addition to the perfusate. Computational models of synaptic, tonic, and evoked currents using a 7-state receptor kinetic model for GABA-ARs were optimized to fit experimental IPSCs and tonic currents and quantitatively define differential properties for synaptic gamma2 subunit- and extrasynaptic delta subunit-containing GABA-ARs for use in subsequent evoked potential models. Evoked potential models were treated as filtered sums of individual IPSCs, with diffusion/spillover to perisynaptic receptors. Simulations then characterized the function and contribution of receptors for different stimulus patterns and conditions associated with or causing seizures. Results: GABA release evoked by hi-frequency PPS increases GABA-AR tonic currents by 13.5 +/- 4.8 pA (p<.05). This non-desensitizing extrasynaptic tonic inhibitory current estimates a 1-2 uM increase in extracellular GABA. Exposure of synaptic GABA-ARs to 3 uM of GABA rapidly desensitizes receptors and diminishes mIPSC amplitude from –51.3 +/- 15.2 to –28.8 +/- 6.7pA (p<.001). Computational models of GABA-ARs optimized to fits of IPSCs show pulsatile stimulation of 20, 40 and 160 Hz for 400 msec duration also reduces IPSC amplitude by 39%, 57% and 75%, respectively. Recovery from desensitization is slow and completely prevented by intermittent activation with frequencies as low as 0.5 to 2 Hz. After 2/20 Hz PPS for 5 min, evoked responses show a ‘late’ component consistent with ‘spillover’ activation of an additional 4 extrasynaptic for every 36 postsynaptic per synapse GABA-ARs. Progressive desensitization of synaptic receptors before slower accumulation of inhibition from extrasynaptic sources occurs with successive stimulations. Conclusions: Initial losses of inhibition through desensitization precede increases from extrasynaptic sources. Although spillover contributes up to 60% of the total inhibitory current, dynamic effects from slowing potentiate synchrony. Shifting patterns of activation of synaptic and extrasynaptic GABA-ARs result in differential effects on postsynaptic receptor desensitization and spillover with impacts on circuit dynamics during seizure evolution or progression to SE. Funding: None