Abstracts

Rationale: The GABA_A 2R43Q mutation confers Generalized Epilepsy with Febrile Seizures Plus (GEFS+) in humans (Wallace et al, 2001) and absence-like seizures in knock-in mice (Tan et al, 2007). In heterologous expression systems this mutation alters receptor kinetics (Goldschen-Ohm et al, 2009), causes deficits in receptor assembly or trafficking (Sancar and Czajkowski, 2004; Hales et al, 2005; Kang et al, 2006), and in particular, alters the expression of 5 subunit-containing receptors (Eug ne et al, 2007) that contribute to nonsynaptic tonic inhibitory currents. Previous work in our lab shows mice (RQ) with this mutation lack tonic inhibitory currents in cortex and thalamus. Here we use Western blots and extracellular multichannel recordings to show that RQ mice display decreased trafficking of the 5-subunit to the membrane surface in cortical neurons, resulting in an increased spontaneous firing rate in vitro.Methods: Brain areas were dissected and fractionated, producing plasma membrane (PM) and organelle (ER) fractions. Western blot analysis was performed for GABA_A receptor 1, 5, 4, , and 2 subunits, and -actin as a loading control. The normalized PM quantity for each subunit was divided by the normalized ER quantity to calculate the PM/ER ratio. These values were compared between wild-type (RR) and RQ (Fig 1). Multichannel recordings were made with two NeuroNexus 16-channel recording arrays placed in the cortex and thalamus of 400 m thalamocortical slices. Long recordings (> 180 min) in low-Mg⁺ consisted of spontaneous neuronal activity. Mean firing rates were calculated for each cell over a 40 minute period before and after the addition of 30uM L655,708, a selective inverse agonist to the GABAA 5-containing receptors, or a vehicle control. All cells from each condition for all experiments were pooled and compared with a Wilcoxon Rank-Sum test and displayed with CDFs (Fig 2).Results: RQ mice cortex showed a decreased PM/ER ratio for 5 compared to RR (mean SEM; RR:3.33 0.37,n=3;RQ:1.59 0.43,n=4; p<0.05). RQ mice also trend towards a decrease in PM/ER ratio for (RR:1.04 0.15,n=3; RQ:0.61 0.09,n=4;p=0.054) and 4 (RR:1.05 0.34,n=3; RQ:0.48 0.10,n=4;p=0.126). There was no change in the ratio (RR:3.23 0.52,n=3; RQ:3.19 0.44,n=4;p=0.95). RR cortical firing rates were not changed from baseline (BL) after vehicle (Hz) (median IQR;BL:0.029 0.061; Vehicle:0.024 0.051;n=130;p=0.4865). RR slices treated with L655,708 showed increased cortical firing rates compared to baseline (BL-L655:0.058 0.120; L655:0.17 0.29;n=122;p<0.001) or RQ (RQ:0.049 0.115;n=135;p<0.00001). RQ slices display an increased cortical firing rate compared to control (RR:0.034 0.073,n=512; RQ:0.049 0.115;n=135;p<0.05).Conclusions: These results show that the R43Q mutation alters trafficking of extra-synaptic receptors to the cell surface in RQ cortical neurons, and that the resulting loss of tonic inhibitory currents in these neurons sparks an increase in spontaneous firing rates.