Abstracts

Rationale: Hypothalamic hamartomas (HH) are benign tumors arising in the ventral hypothalamus associated with treatment-resistant epilepsy. HH are intrinsically epileptogenic for gelastic seizures. The basic cellular and molecular mechanisms responsible for seizure genesis are unknown. Recent work has demonstrated that HH neurons are predominately small (<16 ?m), express glutamic acid decarboxylase (GAD), and demonstrate intrinsic pacemaker-like firing in resected tissue slices. These neurons occur in poorly-defined clusters, which are the dominant microarchitectural feature of this pathology, and may represent the functional unit for ictogenesis. The cytology of these neurons has not been previously studied. We sought to link the previously defined electrophysiological features with single cell microanatomy in order to define the dendritic and projection patterns of small HH neurons and to better understand how these cells contribute to functional networks. Methods: We examined freshly-resected HH tissue from four patients (mean age 11.8 years; 2 females). 350-400 ?m slices were continuously perfused with aCSF and conventional patch-clamp whole-cell microelectrode recordings were conducted under infrared-differential interference contrast microscopy. Electrophysiological data was acquired with a digitizer (DigiData 1322A, Axon Instruments) and analyzed with pClamp 9.1 (Axon Instruments) and Mini Analysis 6 (Synaptosoft). After whole-cell recordings, the neurons were injected with biocytin (Sigma, St. Louis, MO), then stained with 1:1000 Avidin-AF488 (Invitrogen, Carlsbad, CA). Single cell imaging was performed with a Zeiss confocal microscope with z-stacking capabilities. Two-dimensional representations were additionally rendered with the use of a drawing tube. Results: Fifteen neurons were successfully recorded and injected. The cellular phenotypes were diverse, and a classification for all injected neurons has not yet emerged. One consistent phenotype was identified (n = 5), quite likely representing the small HH neuron, based upon the presence of spontaneous firing with microelectrode recording. The electrophysiological features are noted in the Table. The soma was round or globoid, with maximal diameter in the range of 8 - 23 m. These cells are bipolar with more extensive processes arising from one end. An axon could not always be identified. Processes, likely axonal, are often very thin (0.1 m) but could project up to 820 m away from the soma. Dendritic branches have abundant varicosities, and often demonstrate a corkscrew morphology, but have relatively few spines (Figure). Conclusions: These results suggest that the relatively simplistic two-neuron model of HH epileptogenesis may need to be refined: cellular morphology in HH tissue is more diverse than previously anticipated, and the spontaneously firing small HH neuron can be larger than described in prior reports. Small HH neurons also have processes that project up to 800 m from the soma, suggesting that monosynaptic functional networking can include any other neuron within the cluster, but also potentially enable cluster-to-cluster connectivity.