Abstracts

Rationale: Early GABAergic activation and rapid breakdown is an implicated precursor to seizure initiation and propagation. GABA-ARs containing gamma2 subunits mediate phasic inhibitory postsynaptic currents (IPSCs) in hippocampal granule cells in response to brief high concentration transmitter release and rapidly desensitize to low-level tonic or brief hi-frequency pulsatile GABA exposure. Conversely, extrasynaptic GABA-ARs containing delta subunits largely are non-desensitizing, have greater GABA affinity, and are responsible for tonic inhibitory currents in response to mostly stable low concentrations of extracellular GABA, but also detect synaptic ‘spillover.’ With convulsant stimulation and seizure initiation, a loss of synaptic inhibition occurs and an increase in extracellular GABA (1-3 uM) is inferred from tonic current measurements that could impact both synaptic and extrasynaptic GABA-ARs. Methods: Miniature, spontaneous, and evoked/paired-pulse IPSCs as well as GABA-AR mediated tonic currents were recorded from dentate gyrus granule cells in hippocampal slices with visualized whole-cell patch-clamp techniques. 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 the disparate properties of synaptic gamma2-subunit-containing and extrasynaptic delta subunit-containing GABA-ARs. Evoked potential models were treated as filtered sums of individual IPSCs, permitting diffusion/spillover to extrasynaptic receptors. Simulations then characterized the function and contribution of GABA-ARs at different locations for different stimulus patterns and conditions including those causing seizures. Results: Synaptic receptors rapidly desensitize with nearly a 90% loss of inhibition at 160 Hz after only 100 msec, simulating the effects of epileptic ‘fast ripples’. Lower frequencies such as 20 Hz simulating fast-rhythmic activity achieve 40% steady-state desensitization after 400 msec. Recovery from both ‘fast’ and ‘slow’ desensitized receptor states occurs by 10 sec, but superimposed lower frequency activity (0.5 - 2 Hz) and/or low level GABA (< 1uM) sustains the loss of synaptic inhibition by maintaining a significant proportion of postsynaptic GABA-ARs in desensitized states. Composite models including synaptic and extrasynaptic GABA-ARs show that hi-frequency stimulation promotes GABA spillover, and only a few extrasynaptic delta subunit-containing receptors (~ 4 per synapse vs. 36 postsynaptic gamma subunit-containing receptors per synapse) account for up to 60% of the charge transfer of an evoked inhibitory response, prolonging and broadening the spatial extent of synaptically-released GABA and favoring network slowing. At a frequency of 3-6 Hz, 10-20 % of synaptic GABA-ARs remain desensitized sustaining a loss of inhibition while spillover to extrasynaptic receptors supports a synchronous oscillatory response. At higher frequencies above 20 Hz, spillover inhibition summates to act effectively as tonic inhibition and at frequencies lower than 1 Hz, synaptic receptors can recover from desensitization with a restoration of inhibitory tone. Conclusions: Evidence is provided supporting a mechanism of seizure evolution from an initiation phase of fast-rhythmic activity and loss of synaptic inhibition that progresses to slowed synchrony. This involves a dynamic shift of activation from synaptic to extrasynaptic GABA-ARs, and increased spillover to extrasynaptic GABA-ARs during slowed synchrony favors local spread and interaction between adjacent synapses that would facilitate seizure propagation. Funding: No funding