The current study is the first to report (1) the presence of ripples and fast ripples in human subcortical epileptic brain tissue in vitro; (2) spontaneous epileptiform-like discharges, field potentials, and MUA in human HH slices; and (3) the modulation of “pathologic” high-frequency oscillations (i.e., fast ripples), network physiology, and synchrony of emergent activity in human epileptic tissue by L-type calcium channel blockers.
The type(s) of network activity (i.e., HFOs, MUA, and/or field potentials) and conditions (i.e., under normal aCSF or provoked) that critically contribute(s) to the epileptogenicity of brain tissue has yet to be definitively established. HFOs, specifically fast ripples, are purportedly restricted to the seizure-onset zone, have been recorded during interictal events in the mesial temporal lobe and neocortex of patients with epilepsy, and appear at the same time as EEG spikes (Bragin et al., 1999
; Engel et al., 2003
; Urrestarazu et al., 2007
; Schevon et al., 2009
). Ripples occur in the early phase of seizure discharges and are located within and external to the seizure-onset zone (Allen et al., 1992
; Urrestarazu et al., 2007
). Although ripples are thought to be field potentials of summated inhibitory postsynaptic potentials, fast ripples are considered to reflect “field potentials of population spikes” from clusters of bursting neurons (Engel et al., 2009
). Consistent with these findings, HH tissue is intrinsically epileptogenic, and the strong interplay between large excitatory “principal-like” neurons and clusters of local spontaneously firing small neurons creates a cytoarchitecture that is a prime environment to generate HFOs. Therefore, the presence of fast ripples and/or ripples and their association with field potentials may be a critical component of seizure genesis in HH tissue.
In addition to HFOs, spontaneous and induced field potentials also have been recorded in human epileptic tissues with single electrodes, and their occurrence has been associated with interictal and ictal EEG events (Schwartzkroin & Haglund, 1986
). Synchronous sharp field potentials recorded in human epileptic neocortical slices arise from the initial activation of a small group of neurons firing tens of milliseconds before the field potential peak, and this initial local activation is followed by recruitment of additional neuronal groups with a spatial spread ranging from 200–750 μm (Köhling et al., 1998
). The coherent activity of a local group of neurons is purported to initiate epileptiform discharges, and the recurrence of these field potentials has been implicated in reinforcing synchronous synaptic circuitry, thereby laying the foundation for subsequent ictal events (Traub et al., 1996
; Staley & Dudek, 2006
). The spontaneous field potentials in HH tissue under aCSF conditions and the increased number during provocation with 4-AP may indicate greater coherence of several groups of neurons reinforcing pathologic synaptic circuitry and reflect the tissue’s increased capacity to initiate epileptiform events.
The events recorded in each HH slice may reflect interictal activity. These events represent a small fraction of the network activity of the whole tissue in vivo. The constant spontaneous activity may transition into ictal activity and trigger extrahypothalamic propagation in response to provocation or an inciting event that excites the tissue resulting in subsequent change in potassium and calcium fluxes and initiation of calcium signaling cascades. When provoked in vitro, HH tissue activity increased by hundreds to >1,000% as did the synchronicity of events, suggesting that the cytoarchitecture of HH tissue is able to recruit neighboring networks that may support long-range propagation of seizures within HH tissue. When provoked in vivo, the summated and synchronized hyperexcitability may surpass seizure threshold and yield preictal/ictal activity. In patients, interictal spikes arise from synchronous activation of approximately 5–6 cm2
of cortex. Therefore, epileptiform-like activity (both interictal and ictal) in vitro may arise from the emergent orchestrated synchronicity of events throughout the tissue. We speculate that large projection neurons found in HH may relay the seizure activity to subcortical, cortical, and/or limbic regions resulting in the diverse phenotypic seizures types associated with HH (Kerrigan et al., 2005
; Fenoglio et al., 2007
Blockade of calcium channels has been reported to reduce epileptic activity in animal models of epilepsy and in human epileptic tissue (Köhling et al., 1998
; Straub et al., 2000
; Staley & Dudek, 2006
). Reducing calcium entry may yield an anticonvulsant effect by weakening recurrent synapses, thereby reducing spike frequency and the probability of subsequent propagation of epileptiform-like events in vitro. Blockade of L-type calcium channels markedly reduced field potentials in HH tissue under normal aCSF and provoked conditions. In addition, this is the first study to report that blocking L-type calcium channels attenuated HFOs and abolished their association with field potentials in epileptic tissue. Therefore, preventing intracellular calcium flux through L-type calcium channels may limit synchronous depolarizations and reduce/uncouple HFOs from field potentials.
According to our working hypothesis, spontaneous action potential firing of small neurons drives epileptogenic activity of HH tissue (Fenoglio et al., 2007
). MUA detected in the current study may reflect action potential firing of both small and large neurons with complex excitatory and inhibitory efferent projections. This is further supported by the conversion of paired-pulse depression (possibly due to small-neuron–mediated inhibition) to facilitation and increase in evoked MUA after blockade of GABAA
receptors with picrotoxin (20–100 μm
; potentially attributable to the disinhibition of small neurons). One may speculate that the increased activity following nifedipine may reflect activity initiated by the ~10% of large neurons that are insensitive to L-type calcium modulation. Nifedipine may also differentially affect the firing of clusters of small HH neurons, such that disinhibition of adjacent small neurons (via inhibitory collaterals) and of large HH neurons (via GABAergic input) occurs. The enhanced efficacy of nifedipine following provocation may be attributed to the increased efficacy of dihydropyridines in blocking L-type calcium channels under depolarized conditions (Bean, 1984
In summary, the data from the current study suggest that nifedipine attenuates activity differentially under normal aCSF conditions (which may reduce seizure threshold) and under provoked conditions (i.e., during a seizure). Therefore, if critical epileptogenic elements include (1) the association of field potentials with HFOs under basal or provoked conditions; (2) the frequency of field potentials under basal or provoked conditions; (3) MUA during provoked conditions; and/or (4) propagation of events, then blockade of L-type calcium channels may be anticonvulsant in HH tissue and warrants consideration as an antiepileptic treatment for patients with HH.
HH patients typically exhibit seizures that are refractory to traditional anticonvulsant therapies. The identification of a novel molecular target may ultimately provide an effective pharmacologic option for HH patients with intractable epilepsy. The AED history of 14 of 16 patients indicates treatment with AEDs known to modulate both GABAA
receptors and calcium channels (Table S1
; Simeone, 2010
). The present study highlights the complexity of abnormal brain tissue and suggests that a more specific L-type calcium channel blocker may be efficacious in the treatment of seizures associated with HH despite the lack of a consistent effect in other types of epilepsies (see Kulak et al., 2004
). Due to the peripheral side-effects and limited permeability of nifedipine, other L-type calcium channel blockers with more favorable pharmacokinetics and better central nervous system penetration such as amlodipine or nimodipine may be considered. Delineation of the mechanisms of seizure genesis operant in HH lesions and pharmacologic reduction of such activity will hopefully reveal facets of subcortical epileptogenesis that will also be relevant to other brain structures.