Abstracts

Human hypothalamic hamartomas (HH) are associated with precocious puberty, gelastic seizures and severe refractory epilepsy. Emerging evidence suggests that the HH itself plays a pivotal role in this syndrome. [gamma]-aminobutyric acid is the major inhibitory neurotransmitter in the brain and plays an important role in modulation of fast inhibition through GABA[sub]A[/sub] receptors. Abnormal GABA[sub]A[/sub] receptor function is an important mechanism of epileptogenesis in various types of human epilepsy and animal epileptic models. However, it is unknown whether functional GABA[sub]A[/sub] receptors are natively expressed in HH neurons - and if so, what are the functional, pharmacological and molecular properties of these receptors., Acutely enzymatic/mechanical dissociation of HH neurons was performed. Fresh resected HH tissues were immediately placed in ice-cold dissection solution containing (in mM): 136.7 NaCl, 5 KCl, 0.1 NaH[sub]2[/sub]PO[sub]4[/sub], 9.84 HEPES, 16.6 glucose and 21.9 sucrose. After incubation at 22 [plusmn] 1 [deg]C for at least 1 h, tissue sections were treated with papain (4-6 mg/ml at 31 [ordm]C for 50-60 min), and then one tissue fragment was mechanically dissociated. Isolated single HH cells usually maintained function for 2-6 h. Perforated patch-clamp whole-cell recording combined with U-tube drug application was applied to characterize function and pharmacology of GABA[sub]A[/sub] receptors., Under perforated patch-clamp recording in voltage-clamp mode, GABA induced an inward current ([italic]I[/italic][sub]GABA[/sub]) at a holding potential of [ndash]50 mV. The [italic]I[/italic][sub]GABA[/sub] was mimicked by a GABA[sub]A[/sub] receptor agonist (muscimol) and blocked by GABA[sub]A[/sub] receptor antagonist (bicuculline), suggesting that the [italic]I[/italic][sub]GABA[/sub] was mainly mediated through the activation of the GABA[sub]A[/sub] receptor. The concentration-response relationship curve of [italic]I[/italic][sub]GABA[/sub] showed that the EC[sub]50[/sub] and Hill coefficient were 1.3 mM and 0.9, respectively. The current-voltage relationship current was linear at a reversal potential close to zero with a symmetric external and internal chloride concentration. HH neurons exhibited heterogeneity for allosteric modulations. Although most HH neurons presented an enhancement of [italic]I[/italic][sub]GABA[/sub] by pentobarbital and pregnenalone, only [sim]60% of neurons showed potentiation of [italic]I[/italic][sub]GABA[/sub] by diazepam. RT-PCR showed normal GABA[sub]A[/sub] receptor subunit expression in HH tissues., We are the first to describe the functional, pharmacological and molecular properties of the GABA[sub]A[/sub] receptor expressed in human HH. An understanding of functional GABA[sub]A[/sub] receptors in HH neurons provides new insights into the epileptogenesis of gelastic seizures., (Supported by Women[apos]s Board Fund of the Barrow Neurological Institute and NS056104.)