Characteristic Of The Activity Generated By Human Epileptic Neurons

Spontaneous Epileptiform Activity

One important topic in studying human epileptic tissue is whether spontaneous epileptiform network activity is maintained in vitro after tissue excision. Field potential recordings have demonstrated network synchronization as reflected in population spikes (Figure 2). These spontaneous discharges, which resemble epileptiform spikes seen with

FIGURE 2 Spontaneous field potential discharges and associated intracellular responses in human neocortical (A) and subicular (B) tissue in vitro are sensitive to GABAa receptor blockade by bicuculline (bic). A: Field potential discharges (FP) at condensed (bottom) and expanded time scale (top inset) before (control), during (Bic), and after (washout) bicuculline application. Inset demonstrates that, generally, hyperpolarizing membrane potential (MP) fluctuations of single neurons accompany field discharges. B: FP discharges at condensed time scale (bottom) before (control), during (Bic), and after (washout) bicuculline application. Top insets show two different responses of single pyramidal cells (top recording of each inset) related to field discharges (bottom recording of each inset): Most responses (as in A) are hyper-polarizing (inhibited pyramidal), but a fraction of neurons shows depolarizing (excited pyramidal) potentials, at times leading to bursts. (Modified from Kohling et al., 1998b (A), and Cohen et al., 2002 (B), with permission.)

FIGURE 2 Spontaneous field potential discharges and associated intracellular responses in human neocortical (A) and subicular (B) tissue in vitro are sensitive to GABAa receptor blockade by bicuculline (bic). A: Field potential discharges (FP) at condensed (bottom) and expanded time scale (top inset) before (control), during (Bic), and after (washout) bicuculline application. Inset demonstrates that, generally, hyperpolarizing membrane potential (MP) fluctuations of single neurons accompany field discharges. B: FP discharges at condensed time scale (bottom) before (control), during (Bic), and after (washout) bicuculline application. Top insets show two different responses of single pyramidal cells (top recording of each inset) related to field discharges (bottom recording of each inset): Most responses (as in A) are hyper-polarizing (inhibited pyramidal), but a fraction of neurons shows depolarizing (excited pyramidal) potentials, at times leading to bursts. (Modified from Kohling et al., 1998b (A), and Cohen et al., 2002 (B), with permission.)

intracranial EEG recordings, have been found in neocortical (Kohling et al., 1998b, 1999, 2000) and hippocampal preparations (Cohen et al., 2002). Because in the latter study spontaneous events appeared to be triggered by pacemaker neurons, they indeed might reflect intrinsic epileptogenicity of the tissue.

Evoked Epileptiform Discharges as Models of Epileptiform Synchronization

As in animal studies, electrical stimulation, pharmaco-logic manipulations, or changes in the ionic microenviron-

ment disclose epileptiform activity in human tissue maintained in vitro. For example, Masukawa et al. (1989, 1996) reported that 1-Hz repetitive stimulation of the perforant path induces epileptiform afterdischarges in the dentate gyrus. Pharmacologic manipulations such as bath application of the GABAa receptor antagonist bicuculline (10 mM) also lead to short-lasting epileptiform discharges that resemble interictal events, correspond to intracellular bursts of action potentials, and are accompanied by afterdischarges (Avoli and Olivier, 1989; Franck et al., 1995; Hwa et al., 1991; McCormick, 1989; Tasker et al., 1992). Apart from constituting a model of epileptiform activity, these findings

FIGURE 3 Spontaneous synchronous activity induced by 4-aminopyridine (4-AP) in neocortical slices obtained from patients with mesial temporal lobe epilepsy (MTLE) (A) and focal cortical dysplasia (FCD) (B and C). A: Isolated field potentials occur spontaneously in a MTLE slice analyzed with field potential and [K+]o recordings at 1000 mm from the pia. Note that each field potential event is associated with a transient increase in [K+]o. B: Spontaneous field potential discharges recorded in two slices (a and c panels) obtained from two FCD patients; in both cases the activity is characterized by isolated interictal field potentials (asterisks) and sustained epileptiform events resembling ictal discharges (continuous lines). Note also that the onset of the ictal event is associated with the occurrence of a negative field potential (arrow) followed by a slow negative event (arrowhead) leading to ictal discharge oscillations. The different characteristics of the isolated negative field events (1) and of the ictal discharge onset (2) are shown in (b) for three or four graphically superimposed samples obtained from the experiment in (a); note that the isolated interictal events are of lower amplitude compared with those leading to ictogenesis. C: Temporal relation between slow interictal events and ictal discharge onset during 4-AP application to slices from FCD cortex. In (a), histogram of the probability of occurrence of the interictal activity over a period of 50 seconds before ictal onset normalized to epoch 5 seconds before ictal event; data were obtained from 64 epochs recorded in 11 FCD slices. One of these epochs is shown in (b). Arrow points to time zero (i.e., ictal on set); asterisks identify slow interictal events.

FIGURE 3 Spontaneous synchronous activity induced by 4-aminopyridine (4-AP) in neocortical slices obtained from patients with mesial temporal lobe epilepsy (MTLE) (A) and focal cortical dysplasia (FCD) (B and C). A: Isolated field potentials occur spontaneously in a MTLE slice analyzed with field potential and [K+]o recordings at 1000 mm from the pia. Note that each field potential event is associated with a transient increase in [K+]o. B: Spontaneous field potential discharges recorded in two slices (a and c panels) obtained from two FCD patients; in both cases the activity is characterized by isolated interictal field potentials (asterisks) and sustained epileptiform events resembling ictal discharges (continuous lines). Note also that the onset of the ictal event is associated with the occurrence of a negative field potential (arrow) followed by a slow negative event (arrowhead) leading to ictal discharge oscillations. The different characteristics of the isolated negative field events (1) and of the ictal discharge onset (2) are shown in (b) for three or four graphically superimposed samples obtained from the experiment in (a); note that the isolated interictal events are of lower amplitude compared with those leading to ictogenesis. C: Temporal relation between slow interictal events and ictal discharge onset during 4-AP application to slices from FCD cortex. In (a), histogram of the probability of occurrence of the interictal activity over a period of 50 seconds before ictal onset normalized to epoch 5 seconds before ictal event; data were obtained from 64 epochs recorded in 11 FCD slices. One of these epochs is shown in (b). Arrow points to time zero (i.e., ictal on set); asterisks identify slow interictal events.

suggest the presence of GABAA receptor-mediated inhibition within the human neocortical network, along with its ability to control epileptiform synchronization. However, one study using this technique suggested an impaired GABAergic inhibitory system in some hippocampi with dentate abnormalities; in these experiments, bicuculline-induced bursting occurred at a lower drug concentration in epileptic hippocampal tissue with mossy fiber sprouting than in tissue with a relatively "normal" dentate (Franck et al., 1995).

Another important pharmacologic manipulation has involved the application of the K+ channel blocker 4-aminopyridine (4-AP). As in normal animal tissue, 4-AP produces recurrent interictal events in human neocortical slices removed from temporal lobe epilepsy patients (Figure 3A) (Avoli et al., 1994); this tissue is characterized by no

FIGURE 4 Field potential and intracellular characteristics of the synchronous epileptiform activity and of the spreading depression (SD)-like episodes generated by human neocortical slices superfused with Mg2+-free medium. A: Field potential of spontaneous and stimulus-induced (arrow) epileptiform discharges. B: Typical intracellular and field potential activity associated with an ictal-like event. Note that a long-lasting hyperpolarization follows the end of the epilep-tiform discharge. C: intracellular (top trace) and [K+]o (bottom trace) recordings during an epileptiform discharge (a, left portion of the trace) and two SD-like episodes (a, right portion of the trace and b). The SD in (a) was induced by a train of low-frequency (5Hz) stimuli (continuous line).

FIGURE 4 Field potential and intracellular characteristics of the synchronous epileptiform activity and of the spreading depression (SD)-like episodes generated by human neocortical slices superfused with Mg2+-free medium. A: Field potential of spontaneous and stimulus-induced (arrow) epileptiform discharges. B: Typical intracellular and field potential activity associated with an ictal-like event. Note that a long-lasting hyperpolarization follows the end of the epilep-tiform discharge. C: intracellular (top trace) and [K+]o (bottom trace) recordings during an epileptiform discharge (a, left portion of the trace) and two SD-like episodes (a, right portion of the trace and b). The SD in (a) was induced by a train of low-frequency (5Hz) stimuli (continuous line).

obvious structural aberration, including abnormal lamination. In contrast, a similar dose of 4-AP elicits NMDArecep-tor-mediated ictal discharges in neocortical tissue obtained from patients with Taylor's type, focal cortical dysplasia (FCD) (Figure 3B). FCD corresponds to a localized disruption of the normal cortical lamination with an excess of large, dysmorphic neurons (Spreafico et al., 1998; Taylor et al., 1971). These studies (Avoli et al., 1999; D'Antuono et al., 2004) have shown that epileptiform synchronization leading to in vitro ictal activity in the human FCD tissue is initiated by a synchronizing mechanism that paradoxically relies on GABAA receptor activation, causing sizeable increases in [K+]o. Moreover, this mechanism may be facilitated by the decreased ability of GABAB receptors to control GABA release from interneuron terminals.

In addition to the bicuculline and 4-AP, epileptiform discharges are also recorded with field potential and intracel-lular techniques in human neocortical (Avoli et al., 1991, 1995; Kohling et al., 1998b, 1999) and hippocampal slices (Remy et al., 2003) during the application of Mg2+-free bathing medium. These events are often characterized by similarities in duration and waveform to the electrographic seizures recorded in vivo (Figure 4A and B). Moreover, human neocortical slices superfused with Mg2+-free medium generate spreading depression (SD)-like episodes (Figure 4C). SDs have also been recorded during hypoxia (Kohling et al., 1996b, 1998a) and following local local pressure ejection of 3 M KCl (Gorji et al., 2001).

As reported to occur in animal experiments, Mg2+-free epileptiform events recorded in human tissue are readily blocked by NMDA receptor antagonists. Because Mg2+-free-induced epileptiform activity has been considered as a model of therapy-resistant discharges in vitro (Heinemann et al., 1994; Zhang et al., 1995), Mg2+-free-induced discharges have been used to test the efficacy of standard and new antiepileptic drugs in human tissue. These experiments demonstrated that carbamazepine exerts only a moderate antiepileptic action on Mg2+-free-induced epileptiform activity, whereas vigabatrin is highly effective (Musshoff et al., 2000b); retigabine and melatonin also displayed a suppressive action in this model (Fauteck et al., 1995; Straub et al., 2001).

Synaptic Plasticity

Using field potential recordings, several groups have revealed that in epileptic hippocampus, repetitive stimulation leads to disproportionately large responses and after-discharges. Moreover, synaptic depression mediated via group III (but not group II) metabotropic glutamate receptor (mGluR) is impeded in sclerotic but not in nonsclerotic human epileptic hippocampus (Dietrich et al., 1999b, 2002; Masukawa et al., 1989, 1996). Further, studies using paired stimuli in the dentate gyrus to test the strength of recurrent inhibition or presynaptic modulation confirmed that inhibition in epileptic tissue is presumably intact and dependent on presynaptic group II metabotropic glutamate receptors (as in animal tissue; Dietrich et al., 2002; Masukawa et al., 1996; Swanson et al., 1998; Uruno et al., 1995). In these studies, as in other investigations, a distinction between sclerotic and nonsclerotic hippocampus may help to circumvent the problem of lacking appropriate control tissue. As proposed by Dietrich et al. (1999, 2002), sclerotically altered hippocampus can differ from nonsclerotic tissue; for the purpose of comparison, the latter specimens may be viewed as "control" tissue. Lastly, perforant path long-term poten-tiation elicited by high-frequency stimulus trains was also lost in sclerotic but not in nonsclerotic hippocampus (Beck et al., 2000).

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