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  • Small HH neurons have spontaneous pacemaker like firing resu

    2018-11-14

    Small HH neurons have spontaneous pacemaker-like firing resulting from intrinsic membrane properties (Kim do et al., 2008; Wu et al., 2007; Wu et al., 2008; Wu et al., 2005). Most large HH neurons exhibit a functionally immature response to GABA exposure, with depolarization and increased firing, resulting from reversal of the transmembrane chloride potential (Kim do et al., 2008; Kim et al., 2009; Wu et al., 2008). Taken together, these findings suggest a cellular model in which clusters of spontaneously firing interneurons paradoxically excite projection neurons in a functional network (Wu et al., 2015). There is indirect evidence for network activity within HH neuron clusters with microelectrode field recordings of high-frequency oscillations in perfused HH tissue slices (Simeone et al., 2011) and highly synchronous firing of single units with microelectrode field recordings in situ (prior to surgical resection) (Steinmetz et al., 2013). Two basic cellular mechanisms are required for seizure activity—imbalance between excitatory and inhibitory influences with net excitation of the cellular network and enhanced synchrony of neuronal firing (Avoli et al., 2005). We hypothesized that synchrony of firing activity within HH tissue is mediated in part by non-synaptic mechanisms, specifically gap junctions, which are capable of electrically linking adjacent neurons. In this study, we tested our hypothesis using multiple experimental approaches. The MAPK Inhibitor Library microscopy demonstrated an evidence of existence of gap junctions between small-sized GABA neurons in HH slices. The functional gap junctions between GABA neurons in HH slices were supported by microinjection of small HH neurons with biocytin via recording electrode under patch-clamp recording conditions demonstrates dye-coupling of adjacent neurons. Although the gap junctions are known to express in normal hypothalamic tissues (Condorelli et al., 2000), our Western-blot data showed a significant increase in expression level of Cx36 and Cx43 in HH tissue compared to normal control adjacent mammillary body tissue. These data suggest a possibility that enhanced gap junction proteins may promote GABA neuron synchronization and form the seizure-like discharges in HH lesion. This idea was further supported by electrophysiological experiments, in which, pharmacological blockade of gap junctions abolishes seizure-like discharges in freshly resected, perfused HH tissue slices without suppressing spontaneous single-unit firing activity. Our findings demonstrate the presence of neuronal gap junctions and suggest that gap junctions have a mechanistic role in generating seizure activity in HH tissue. We acknowledge technical limitations to this study. We have utilized age-matched human autopsy hypothalamic tissue (mammillary body) as a control tissue for Western blots. We recommend mammillary body for control studies as this nucleus is immediately adjacent to all HH lesions associated with epilepsy (Parvizi et al., 2011) and it is readily identifiable in autopsy specimens. However, the embryological relationship between mammillary body and HH is unexplored and the cellular phenotypes are likely dissimilar. The increase of both Cx36 and Cx43 may relate to immature properties of HH tissue, as reflected by functional immaturity of large HH neurons with paradoxical excitation in response to GABA ligands. For our electrophysiological study with gap junction blockers, all comparisons were set up in the same recorded slice (or cell) before during and after gap junction blocker exposure. Another limitation is that we were not able to do in vivo study directly in HH patients in this paper. However, the intrinsic epileptic feature of HH allows us to test the role of gap junctions in epileptogenesis using HH slices, which provides significant insights into understanding of the impact of gap junctions in gelastic seizure genesis. While providing new insights into the cellular mechanisms responsible for gelastic seizures arising in HH tissue, these results also suggest a novel approach to treatment, prompting research to evaluate the safety and efficacy of gap-junction blockers in HH patients. Gap-junction blockers are currently under investigation in people with migraines; the proposed mechanism in those studies is to attenuate or abolish cortical-spreading depression (Silberstein, 2009). Clear evidence for efficacy is thus far lacking, but gap-junction blockers appear to be safe and well tolerated. Based upon the findings MAPK Inhibitor Library reported here, we propose that gap-junction blockers are worthy of further investigation for treating seizures associated with HH and other types of epilepsy.