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Non-technical summary The inhibitory neurotransmitter GABA activates two distinct receptors of which the GABA A receptor is mainly a Cl − conducting ion channel. The proper functioning of inhibition at the GABA A receptor depends on the ionic gradient prevailing during receptor activation, and in epilepsy there is an aberrant Cl − gradient. Using rat and human cortical neurones and pharmacological inhibitors, we calculated the contributions of neuronal Cl − extrusion by the Na + -K + -2Cl − transporter NKCC1, the K + -coupled Cl − transporter KCC2 and the voltage-gated Cl − channel ClC2. We found that KCC2 is the major route of Cl − extrusion and that reduced KCC2 Cl − extrusion is likely to be the initial step of disturbed Cl − regulation. The results contribute to our understanding of epilepsy.Abstract Considerable evidence indicates disturbances in the ionic gradient of GABA A receptor-mediated inhibition of neurones in human epileptogenic tissues. Two contending mechanisms have been proposed, reduced outward and increased inward Cl − transporters. We investigated the properties of Cl − transport in human and rat neocortical neurones (layer II/III) using intracellular recordings in slices of cortical tissue. We measured the alterations in reversal potential of the pharmacologically isolated inhibitory postsynaptic potential mediated by GABA A receptors (IPSP A ) to estimate the ionic gradient and kinetics of Cl − efflux after Cl − injections before and during application of selected blockers of Cl − routes (furosemide, bumetanide, 9-anthracene carboxylic acid and Cs + ). Neurones from human epileptogenic cortex exhibited a fairly depolarized reversal potential of GABA A receptor-mediated inhibition (E IPSP−A ) of −61.9 ± 8.3 mV. In about half of the neurones, the E IPSP-A averaged −55.2 ± 5.7 mV, in the other half, 68.6 ± 2.3 mV, similar to rat neurones (−68.9 ± 2.6 mV). After injections of Cl − , IPSP A recovered in human neurones with an average time constant (τ) of 19.0 ± 9.6 s (rat neurones: 7.2 ± 2.4 s). We calculated Cl − extrusion rates (1/τ) via individual routes from the τ values obtained in different experimental conditions, revealing that, for example, the K + -coupled Cl − transporter KCC2 comprises 45.3% of the total rate in rat neurones. In human neurones, the total rate of Cl − extrusion was 63.9% smaller, and rates via KCC2, the Na + -K + -2Cl − transporter NKCC1 and the voltage-gated Cl − channel ClC were smaller than in rat neurones by 80.0%, 61.7% and 79.9%, respectively. The rate via anion exchangers conversely was 14.4% larger in human than in rat neurones. We propose that (i) KCC2 is the major route of Cl neurones, (ii) reduced KCC2 is the initial step of disturbed Cl − regulation and (iii) reductions in KCC2 contribute to depolarizing E IPSP-A of neurones in human epileptogenic neocortex.

Anomalous hippocampal inhibition is involved in temporal lobe epilepsy, and reduced gephyrin immunoreactivity in the temporal lobe epilepsy hippocampus has been reported recently. However, the mechanisms responsible for curtailing postsynaptic gephyrin scaffolds are poorly understood. Here, we have investigated gephyrin expression in the hippocampus of patients with intractable temporal lobe epilepsy. Immunohistochemical and western blot analyses revealed irregular gephyrin expression in the cornu ammonis of patients with temporal lobe epilepsy and four abnormally spliced gephyrins lacking several exons in their G-domains were isolated. Identified temporal lobe epilepsy gephyrins have oligomerization deficits and they curtail hippocampal postsynaptic gephyrin and GABA(A) receptor α2 while interacting with regularly spliced gephyrins. We found that cellular stress (alkalosis and hyperthermia) induces exon skipping in gephyrin messenger RNA, which is responsible for curtailed postsynaptic gephyrin and GABA(A) receptor α2 scaffolds. Accordingly, we did not obtain evidence for gephyrin gene mutations in patients with temporal lobe epilepsy. Cellular stress such as alkalosis, for example arising from seizure activity, could thus facilitate the development of temporal lobe epilepsy by reducing GABA(A) receptor α2-mediated hippocampal synaptic transmission selectively in the cornu ammonis.

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SUMMARYPurpose: Hyperpolarization-activated cation currents (I H ) play a pivotal role in the control of neuronal excitability. In animal models of epilepsy both increases and decreases of I H have been reported. We, therefore, characterized properties of I H in human epileptogenic neocortex. Methods: Layer II/III neurons in slices from epilepsy surgery tissues and rat cortex were investigated with whole-cell patch-clamp recordings. Results: A total of 484 neurons from 96 temporal lobe epilepsy (TLE) tissues and 32 neurons from 8 frontal lobe epilepsy (FLE) tissues were recorded. Voltage-clamp recordings revealed on hyperpolarizing command steps two time-and voltagedependent inward currents, namely a fast, Ba 2+ -sensitive current (K IR ) and a slowly activating current, namely consisting of two kinetically distinct components sensitive to the established I H blocker ZD7288. Only, the fast component (I H (fast)) of TLE neurons was on average smaller and activated more slowly (density 2.7 ± 1.6 pA/ pF; tau 38.4 ± 34.0 ms) than in FLE neurons (4.7 ± 2.3 pA/pF; 16.6 ± 7.9 ms; p < 0.001 for both). Within the TLE tissues the I H (fast) density (averaged per patient) was smaller in cases with numerous annual grand mal seizures (GM; 2.2 ± 0.6 pA/pF) compared to those with few GM (2.8 ± 1.0 pA/pF; p = 0.0184). A similar difference was obtained in the case of complex partial seizures (CPS; many CPS 2.2 ± 0.6 pA/pF; few CPS 2.9 ± 1.0 pA/pF, p = 0.0037). Discussion: The biophysical properties of I H in cortices from TLE, FLE, and rat tissue suggest a deficit of HCN1 subunits in the human epileptogenic neocortex, which in turn may increase excitability and probability of seizure activity.