Abstracts

Rationale: The human hypothalamic hamartoma (HH) is a rare epileptogenic lesion associated with medically refractory gelastic seizures. Although the underlying mechanisms of epileptogenesis in HH lesions remain unclear, we have recently reported that activation of GABA_A receptors in HH brain slices results in neuronal excitation through chloride efflux, which secondarily activates L-type voltage-gated calcium channels (Kim et al, Epilepsia 2008). Previous studies have implicated the bicarbonate (HCO3^-) as well the chloride ion in GABA_A receptor-mediated depolarization. In the present study, we asked whether HCO3^- may contribute to GABA-induced excitation in surgically-resected human HH tissue. Methods: HH brain slices (300 µm) were acutely prepared from small tissue blocs taken from HH patients (N=7) undergoing either endoscopic or transcallosal approaches toward surgical resection, and studied using standard cellular electrophysiological and fura-2AM ratiometric calcium imaging techniques. Results: In gramicidin-perforated patch recordings using HCO3^--containing saline, the GABA_A receptor agonist muscimol (30 µM) provoked membrane depolarization in over 70% of large (18-24 µM) non-spontaneous firing HH neurons (N=31 cells from 7 cases). Subsequent perfusion of HCO3^--free HEPES buffer for 10 min prior to muscimol application (which reportedly depletes intracellular HCO3^-; Dallwig et al, Eur J Physiol 1999) resulted in partial suppression of membrane depolarization. This suppressive effect was also seen in large HH neurons that depolarized in response to muscimol (with partial, full and no effects seen in 35%, 29% and 35% tested neurons, respectively). Consistent with our previous report (Kim et al, Epilepsia 2008), we found that the L-type calcium channel blocker nifedipine (100 µM) strongly prevented muscimol-induced HH neuronal excitation and fully inhibited intracellular calcium accumulation. Upon pre-incubation with HCO3^--free HEPES buffer (and in the absence of nifedipine), approximately 60% of large HH neurons tested exhibited a significant reduction in calcium accumulation induced by muscimol. Conclusions: Collectively, our results indicate that the bicarbonate ion is involved in muscimol-induced neuronal excitation in the majority of large HH neurons, and suggest that efflux of bicarbonate through GABA_A receptors contributes to the shift of E_GABA toward a more depolarized resting membrane potential. Together with chloride efflux through GABA_A receptors, bicarbonate-mediated membrane depolarization results in subsequent activation of L-type calcium channels.(Supported by NIH grant NS057786 and the Barrow Neurological Foundation.)