The role of neuronal nicotinic receptors (nAChRs) in the pathogenesis of autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE): a study on wild-type and conditional transgenic mice expressing the beta2-V287L subunit

We studied mice conditionally expressing the beta2-V287L nAChR subunit, linked to ADNFLE. Expression of beta2-V287L during the first two postnatal weeks (a critical phase of synaptic maturation) is necessary for the epileptic phenotype to manifest. MATURE NEOCORTEX. Compared to the control littermates (CTRL), adult mice (P60) expressing beta2-V287L (TG) display no alteration in neocortex thickness, neuron count, nAChR density, and expression of GABAergic markers. Densitometric analyses of KCC2 (the major Cl- extruder) displayed increased expression in prefrontal (PFC) layer V and a decrease in the thalamic reticular nucleus, in TG mice. In pyramidal neurons of PFC layer V, we tested the effect of 10 microM nicotine on spontaneous excitatory (EPSC) and inhibitory (IPSC) postsynaptic currents. The basal EPSC/IPSC frequency ratio was similar in TG and CTRL, but nicotine tripled the EPSC frequency in TG mice, an effect stronger than observed in CTRL. Hence, beta2-V287L mainly augmented the glutamatergic activity. Pharmacological tests confirmed the role of alpha4beta2 nAChRs. The miniature postsynaptic events and the EPSC amplitudes show that most of the effect occurred on synaptic terminals. TG mice were also more susceptible to develop epileptiform activity in the presence of low Mg2+ and 4AP. We are now investigating how ACh regulates the basal hyperexcitability. DEVELOPMENT. Because of its role in network maturation, we studied the developmental balance between the expression of KCC2 and NKCC1 (which absorbes Cl-). The kinetics of transporters’ expression was not altered by expression of beta2-V287L. Consistently, the timing of developmental switch of the GABAAR reversal potential was identical in TG and CTRL mice. However, studying the EPSCs revealed an approximate triplication of the basal glutamatergic activity in TG mice, in the second postnatal week. We conclude that the beta2-V287L is not directly implicated in the maturation of the GABAergic system, but causes an early increase in the efficacy of the glutamatergic transmission, in agreement with the results obtained in the mature PFC. IN VITRO MODEL OF ADNFLE. By multi-electrode array (MEA) methods, we found that longterm TG neocortical cultures develop long (10–32 s) global bursting events, whose duration is similar to the in vivo seizures. No pretreatment with convulsants was necessary, thus reproducing the features of genetic epilepsies. The long bursts were blocked by carbamazepine, a drug of choice in ADNFLE. CONCLUSIONS. Beta2-V287L increased the background excitability and the sensitivity to nAChR agonists, which we attribute to increase of glutamatergic transmission during early postnatal stages, as well as intrinsic hyperfunctionality of alpha4beta2-V287L. Our chronic model of hyperexcitability opens the way to in vitro studies on the role of beta2-V287L on excitatory synapse formation and its modulation by antiepileptics.