Abstracts

Rationale: The hypothalamic hamartoma (HH) is a rare human brain malformation associated with gelastic seizures. Seizures are known to originate from the HH lesion itself, but the underlying mechanisms are unknown. Recent studies have implicated a pathophysiological role for the chloride extruding and chloride importing co-transporters NKCC1 and KCC2, respectively, in seizure genesis (Science 2002; 298:5597; Epilepsia 2007; 48:4:663). Specifically, high NKCC1/KCC2 ratios favor a depolarizing response to GABA_A receptor activation. Further, it is known that GABA-induced chloride efflux triggers calcium influx through voltage-dependent calcium channels (VDCCs) in the immature brain (J Physiol 2004; 557:829). Thus, we asked whether activation of GABA_A receptors on HH neurons in freshly resected HH tissue slices results in neuronal excitation, and if this phenomenon involves altered expression of NKCC1/KCC2 and/or voltage-dependent calcium channels (VDCCs).Methods: Routine cellular electrophysiological (gramicidin perforated patch), ratiometric calcium (fura-2AM) imaging and immunocytochemical (antibodies directed against NKCC1, KCC2, and CaV1.2 & CaV1.3 L-type calcium channel subunits) techniques were used to examine the effects of muscimol (30 μM) on small (~10 μm) spontaneously firing GABAergic neurons and large (~20 μm) quiescent HH neurons (see Epilepsia 46:Suppl.8: 3.006).Results: Most (70%) large neurons (but none of the small cells) in HH brain slices depolarized after muscimol (30 μM) application; these responses were suppressed by the GABA_A receptor antagonist bicuculline (50 μM). Furthermore, large depolarizing neurons had a more positive reversal potential (E_muscimol) and significantly higher intracellular chloride concentrations compared to those that hyperpolarized in response to muscimol. Immunolabeling experiments revealed co-expression of the chloride co-transporters NKCC1 and KCC2 in 65% of large neurons, whereas 23% and 11% of large cells expressed solely NKCC1 and KCC2, respectively. Pharmacological blockade of NKCC1 with bumetanide (20 μM) only partially suppressed muscimol-induced excitation in large neurons. Muscimol induced a significant rise in intracellular calcium in large depolarizing neurons. Concordant with robust expression of CaV1.2 and CaV1.3 subunits, the L-type calcium channel blocker nifedipine (100 μM) prevented muscimol-induced neuronal excitation. Interestingly, application of nifedipine also abolished the spontaneous firing of small HH neurons.Conclusions: Our data suggest that GABA_A receptor-mediated excitation, arising in part from differential expression of NKCC1 and KCC2, and resulting in activation of L-type calcium channels, may contribute to seizure genesis in HH tissue. Moreover, given the ready availability of L-type calcium channel blockers, these results have strong clinical ramifications for the treatment of seizures associated with HH lesions (Supported by NIH grant NS 044846 and the Barrow Neurological Foundation).