GABAA receptor-mediated excitation in dissociated neurons from human hypothalamic hamartomas

Abstract

The cellular mechanisms underlying intrinsic epileptogenesis in human hypothalamic hamartoma (HH) are unknown. We previously reported that HH tissue is composed predominantly of GABAergic neurons, but how GABAergic-neuron-rich HH tissue is intrinsically epileptogenic is unclear. Here, we tested the hypotheses that some HH neurons exhibit immature features and that GABA excites these neurons via activation of GABAA receptors (GABAARs). Gramicidin-perforated and cell-attached patch-clamp recordings were performed using freshly-dissociated HH neurons to evaluate GABAAR-mediated currents, Cl− equilibrium potentials, and intracellular Cl− concentrations. Single-cell RT-PCR and immunocytochemical techniques were used to examine cation-Cl− co-transporter (NKCC1 and KCC2) gene and KCC2 protein expression and molecular markers of maturation. From a total of 93 acutely-dissociated HH neurons from 34 patients, 76% were small (soma: 6–9 μm) and 24% were large (soma: >20 µm) in size. Under gramicidin-perforated patch recording conditions, GABAAR activation depolarized/excited large but hyperpolarized/inhibited small HH neurons in most cases. Compared to small HH neurons, large HH neurons exhibited more positive Cl− equilibrium potentials, higher intracellular Cl− concentrations, lower KCC2 expression, and an immature phenotype, consistent with GABAAR-mediated excitation. Taken collectively, we provide novel evidence for and mechanistic insights into HH epileptogenicity: GABAAR-mediated excitation.