Abstracts

Rationale: Human hypothalamic hamartomas (HH) are rare, intrinsically epileptogenic, developmental malformations. Recently, we reported that HH lesions contain two predominant types of neurons, small- and large-sized, and that the majority of small HH neurons are GABAergic and show spontaneous, intrinsic pacemaker-like firing activity.1,2 However, the mechanisms involved in epileptogenesis remain unknown.Methods: Here we tested the hypothesis that GABAA receptor-mediated excitation occurs in some HH neurons, contributing to inherent epileptogenesis. Gramicidin-perforated and cell-attached recordings in acutely dissociated HH neurons were applied, and cell and molecular biological approaches were combined.Results: Gramicidin-perforated and cell-attached recordings demonstrated that the GABAA receptor agonist, muscimol, excited (depolarized) large HH neurons, but inhibited (hyperpolarized) small HH neurons in most cases. Based on current-voltage relationships for GABA responses, the estimated intracellular Cl- concentrations were 39.1 ± 2.8 and 19.2 ± 3.7 mM in large and small neurons, respectively (p<0.01). Single-cell RT-PCR and immunostaining studies revealed lower expression of the chloride-extruding, K+-Cl- cotransporter (KCC2) in large HH neurons. Microtransplantation of HH or control (normal human hypothalamic) tissue membrane fractions into Xenopus oocytes produced cells showing GABA-mediated whole-cell current responses with Cl- equilibrium potentials of -13.2 ± 1.3 or -23.9 ± 0.8 mV (p<0.001), respectively, further associating HH tissues with elevated internal Cl- concentrations.Conclusions: Collectively, these results suggest that GABAA receptor signaling on most large acutely-dissociated HH neurons, and perhaps in HH tissue, is excitatory rather than inhibitory due to low expression of KCC2 and abnormally elevated internal Cl- levels. The immature profile for Cl- distribution, KCC2 expression, and GABAA receptor excitatory activity may play important roles in HH epileptogenicity.