Repetitive low-frequency stimulation reduces epileptiform synchronization in limbic neuronal networks.

Deep-brain electrical or transcranial magnetic stimulation may represent a therapeutic tool for controlling seizures in patients presenting with epileptic disorders resistant to antiepileptic drugs. In keeping with this clinical evidence, we have reported that repetitive electrical stimuli delivered at approximately 1 Hz in mouse hippocampus-entorhinal cortex (EC) slices depress the EC ability to generate ictal activity induced by the application of 4-aminopyridine (4AP) or Mg2+-free medium (Barbarosie, M., Avoli, M., 1997. CA3-driven hippocampal–entorhinal loop controls rather than sustains in vitro limbic seizures. J. Neurosci. 17, 9308–9314.). Here, we confirmed a similar control mechanism in rat brain slices analyzed with field potential recordings during 4AP (50 μM) treatment. In addition, we used intrinsic optical signal (IOS) recordings to quantify the intensity and spatial characteristics of this inhibitory influence. IOSs reflect the changes in light transmittance throughout the entire extent of the slice, and are thus reliable markers of limbic network epileptiform synchronization. First, we found that in the presence of 4AP, the IOS increases, induced by a train of electrical stimuli (10 Hz for 1 s) or by recurrent, single-shock stimulation delivered at 0.05 Hz in the deep EC layers, are reduced in intensity and area size by low-frequency (1 Hz), repetitive stimulation of the subiculum; these effects were observed in all limbic areas contained in the slice. Second, by testing the effects induced by repetitive subicular stimulation at 0.2–10 Hz, we identified maximal efficacy when repetitive stimuli are delivered at 1 Hz. Finally, we discovered that similar, but slightly less pronounced, inhibitory effects occur when repetitive stimuli at 1 Hz are delivered in the EC, suggesting that the reduction of IOSs seen during repetitive stimulation is pathway dependent as well as activity dependent. Thus, the activation of limbic networks at low frequency reduces the intensity and spatial extent of the IOS changes that accompany ictal synchronization in an in vitro slice preparation. This conclusion supports the view that repetitive stimulation may represent a potential therapeutic tool for controlling seizures in patients with pharmacoresistant epileptic disorders

Lacosamide: a new approach to target voltage-gated sodium currents in epileptic disorders

The mechanism of action of several antiepileptic drugs (AEDs) rests on their ability tomodulate the activity of voltage-gated sodium currents that are responsible for fast action potentialgeneration. Recent data indicate that lacosamide - a compound with analgesic and anticonvulsanteffects in animal models - shares a similar mechanism. When compared with other AEDs, lacosamidehas the unique ability to interact with sodium channel slow inactivation without affecting fastinactivation. This article reviews these findings and discusses their relevance within the context ofneuronal activity seen during epileptiform discharges generated by limbic neuronal networks in thepresence of chemical convulsants. These seizure-like events are characterized by sustained dischargesof sodium-dependent action potentials supported by robust depolarizations thus providingsynchronization within neuronal networks. Generally, AEDs such as phenytoin, carbamazepine andlamotrigine block sodium channels when activated. By contrasts, lacosamide facilitates slowinactivation of sodium channels both in term of kinetics and voltage-dependency. This effect may berelatively selective for repeatedly depolarized neurons such as those participating in seizure activity inwhich the persistence of sodium currents is more pronounced and promotes neuronal excitation. Theclinical effectiveness of lacosamide has been demonstrated in randomized placebo-controlled doubleblindparallel-group, adjunctive-therapy trials in patients with refractory partial seizures. Furtherstudies should determine whether lacosamide effects in animal models and in clinical settings are fullyexplained by its selective action on sodium current slow inactivation or whether other effects (e.g.,interactions with the collapsin-response mediator protein 2) play a contributory role

Impaired Activation of CA3 Pyramidal Neurons in the Epileptic Hippocampus.

We employed in vitro and ex vivo imaging tools to characterize the function of limbic neuron networks in pilocarpine-treated and age-matched, nonepileptic control (NEC) rats. Pilocarpine-treated animals represent an established model of mesial temporal lobe epilepsy. Intrinsic optical signal (IOS) analysis of hippocampal-entorhinal cortex (EC) slices obtained from epileptic rats 3 wk after pilocarpine-induced status epilepticus (SE) revealed hyperexcitability in many limbic areas, but not in CA3 and medial EC layer III. By visualizing immunopositivity for FosB/DeltaFosB-related proteins which accumulate in the nuclei of neurons activated by seizures we found that: (1) 24 h after SE, FosB/DeltaFosB immunoreactivity was absent in medial EC layer III, but abundant in dentate gyrus, hippocampus proper (including CA3) and subiculum; (2) FosB/DeltaFosB levels progressively diminished 3 and 7 d after SE, whereas remaining elevated (p < 0.01) in subiculum; (3) FosB/DeltaFosB levels sharply increased 2 wk after SE (and remained elevated up to 3 wk) in dentate gyrus and in most of the other areas but not in CA3. A conspicuous neuronal damage was noticed in medial EC layer III, whereas hippocampus was more preserved. IOS analysis of the stimulus-induced responses in slices 3 wk after SE demonstrated that IOSs in CA3 were lower (p < 0.05) than in NEC slices following dentate gyrus stimulation, but not when stimuli were delivered in CA3. These findings indicate that CA3 networks are hypoactive in comparison with other epileptic limbic areas. We propose that this feature may affect the ability of hippocampal outputs to control epileptiform synchronization in EC

Diminished presynaptic GABA(B) receptor function in the neocortex of a genetic model of absence epilepsy

Changes in GABA(B) receptor subunit expression have been recently reported in the neocortexof epileptic WAG/Rij rats that are genetically prone to experience absence seizures.These alterations may lead to hyperexcitability by downregulating the function of presynapticGABA(B) receptors in neocortical networks as suggested by a reduction in paired-pulsedepression. Here, we tested further this hypothesis by analyzing the effects induced by theGABA(B) receptor agonist baclofen (0.1-10 μM) on the inhibitory events recorded in vitro fromneocortical slices obtained from epileptic (>180 day-old) WAG/Rij and age-matched, nonepilepticcontrol (NEC) rats. We found that higher doses of baclofen were required todepress pharmacologically isolated, stimulus-induced IPSPs generated by WAG/Rij neuronsas compared to NEC. We also obtained similar evidence by comparing the effects ofbaclofen on the rate of occurrence of synchronous GABAergic events recorded by WAG/Rijand NEC neocortical slices treated with 4-aminopyridine+glutamatergic receptor antagonists.In conclusion, these data highlight a decreased function of presynaptic GABA(B) receptorsin the WAG/Rij rat neocortex. We propose that this alteration may contribute toneocortical hyperexcitability and thus to absence seizures

Selective changes in inhibition as determinants for limited hyperexcitability in the insular cortex of epileptic rats

The insular cortex (IC) is involved in the generalization of epileptic discharges in temporal lobe epilepsy (TLE), while seizures originating in IC can mimic the epileptic phenotype seen in some TLE patients. Few studies have however addressed the changes occurring in the IC in TLE animal models. Here, we analyzed the immunohistochemical and electrophysiological properties of IC networks in non-epileptic control and pilocarpine-treated epileptic rats. Neurons identified with a neuron-specific nuclear protein antibody showed similar counts in the two types of tissue but parvalbumin- and neuropeptide Y-positive interneurons were significantly decreased (parvalbumin, approx. -35%; neuropeptide Y, approx. -38%; P<0.01) in the epileptic IC. Non-adapting neurons were more frequently seen in the epileptic IC during intracellular injection of depolarizing current pulses. In addition, single-shock electrical stimuli elicited network-driven epileptiform responses in 87% of epileptic and in 22% of non-epileptic control neurons (P<0.01) but spontaneous postsynaptic potentials had similar amplitude, duration and intervals of occurrence in the two groups. Finally, pharmacologically isolated, GABAA receptor-mediated inhibitory postsynaptic potentials had more negative reversal potential (P<0.01) and higher peak conductance (P<0.05) in epileptic tissue. These data reveal moderate increased network excitability in the IC of pilocarpine-treated epileptic rats. We propose that such limited degree of hyperexcitability originates from loss of parvalbumin- and neuropeptide Y-positive interneurons that is compensated by an increased drive for GABAA receptor-mediated inhibition

Reduced GABA(B) receptor subunit expression and paired-pulse depression in a genetic model of absence seizures

Neocortical networks play a major role in the genesis of generalized spike-and-wave (SW) discharges associated with absence seizures in humans and in animal models, including genetically predisposed WAG/Rij rats. Here, we tested the hypothesis that alterations in GABAB receptors contribute to neocortical hyperexcitability in these animals. By using Real-Time PCR we found that mRNA levels for most GABAB(1) subunits are diminished in epileptic WAG/Rij neocortex as compared with age-matched non-epileptic controls (NEC), whereas GABAB(2) mRNA is unchanged. Next, we investigated the cellular distribution of GABAB(1) and GABAB(2) subunits by confocal microscopy and discovered that GABAB(1) subunits fail to localize in the distal dendrites of WAG/Rij neocortical pyramidal cells. Intracellular recordings from neocortical cells in an in vitro slice preparation demonstrated reduced paired-pulse depression of pharmacologically isolated excitatory and inhibitory responses in epileptic WAG/Rij rats as compared with NECs; moreover, paired-pulse depression in NEC slices was diminished by a GABAB receptor antagonist to a greater extent than in WAG/Rij rats further suggesting GABAB receptor dysfunction. In conclusion, our data identify changes in GABAB receptor subunit expression and distribution along with decreased paired-pulse depression in epileptic WAG/Rij rat neocortex. We propose that these alterations may contribute to neocorticalhyperexcitability and thus to SW generation in absence epilepsy

GABA-enhanced collective behavior in neuronal axons underlies persistent gamma-frequency oscillations

Gamma (30–80 Hz) oscillations occur in mammalian electroencephalogram in a manner that indicates cognitive relevance. In vitro models of gamma oscillations demonstrate two forms of oscillation: one occurring transiently and driven by discrete afferent input and the second occurring persistently in response to activation of excitatory metabotropic receptors. The mechanism underlying persistent gamma oscillations has been suggested to involve gap-junctional communication between axons of principal neurons, but the precise relationship between this neuronal activity and the gamma oscillation has remained elusive. Here we demonstrate that gamma oscillations coexist with high-frequency oscillations (>90 Hz). High-frequency oscillations can be generated in the axonal plexus even when it is physically isolated from pyramidal cell bodies. They were enhanced in networks by nonsomatic -aminobutyric acid type A (GABAA) receptor activation, were modulated by perisomatic GABAA receptor-mediated synaptic input to principal cells, and provided the phasic input to interneurons required to generate persistent gamma-frequency oscillations. The data suggest that high-frequency oscillations occurred as a consequence of random activity within the axonal plexus. Interneurons provide a mechanism by which this random activity is both amplified and organized into a coherent network rhythm

Anti-epileptic effect of Ganoderma lucidum polysaccharides by inhibition of intracellular calcium accumulation and stimulation of expression of CaMKII a in epileptic hippocampal neurons

Purpose: To investigate the mechanism of the anti-epileptic effect of Ganoderma lucidum polysaccharides (GLP), the changes of intracellular calcium and CaMK II a expression in a model of epileptic neurons were investigated. Method: Primary hippocampal neurons were divided into: 1) Control group, neurons were cultured with Neurobasal medium, for 3 hours; 2) Model group I: neurons were incubated with Mg2+ free medium for 3 hours; 3) Model group II: neurons were incubated with Mg2+ free medium for 3 hours then cultured with the normal medium for a further 3 hours; 4) GLP group I: neurons were incubated with Mg2+ free medium containing GLP (0.375 mg/ml) for 3 hours; 5) GLP group II: neurons were incubated with Mg2+ free medium for 3 hours then cultured with a normal culture medium containing GLP for a further 3 hours. The CaMK II a protein expression was assessed by Western-blot. Ca2+ turnover in neurons was assessed using Fluo-3/AM which was added into the replacement medium and Ca2+ turnover was observed under a laser scanning confocal microscope. Results: The CaMK II a expression in the model groups was less than in the control groups, however, in the GLP groups, it was higher than that observed in the model group. Ca2+ fluorescence intensity in GLP group I was significantly lower than that in model group I after 30 seconds, while in GLP group II, it was reduced significantly compared to model group II after 5 minutes. Conclusion: GLP may inhibit calcium overload and promote CaMK II a expression to protect epileptic neuron

Facilitation of epileptic activity during sleep is mediated by high amplitude slow waves

Epileptic discharges in focal epilepsy are frequently activated during non-rapid eye movement sleep. Sleep slow waves are present during this stage and have been shown to include a deactivated ('down', hyperpolarized) and an activated state ('up', depolarized). The 'up' state enhances physiological rhythms, and we hypothesize that sleep slow waves and particularly the 'up' state are the specific components of non-rapid eye movement sleep that mediate the activation of epileptic activity. We investigated eight patients with pharmaco-resistant focal epilepsies who underwent combined scalp-intracerebral electroencephalography for diagnostic evaluation. We analysed 259 frontal electroencephalographic channels, and manually marked 442 epileptic spikes and 8487 high frequency oscillations during high amplitude widespread slow waves, and during matched control segments with low amplitude widespread slow waves, non-widespread slow waves or no slow waves selected during the same sleep stages (total duration of slow wave and control segments: 49 min each). During the slow waves, spikes and high frequency oscillations were more frequent than during control segments (79% of spikes during slow waves and 65% of high frequency oscillations, both P ~ 0). The spike and high frequency oscillation density also increased for higher amplitude slow waves. We compared the density of spikes and high frequency oscillations between the 'up' and 'down' states. Spike and high frequency oscillation density was highest during the transition from the 'up' to the 'down' state. Interestingly, high frequency oscillations in channels with normal activity expressed a different peak at the transition from the 'down' to the 'up' state. These results show that the apparent activation of epileptic discharges by non-rapid eye movement sleep is not a state-dependent phenomenon but is predominantly associated with specific events, the high amplitude widespread slow waves that are frequent, but not continuous, during this state of sleep. Both epileptic spikes and high frequency oscillations do not predominate, like physiological activity, during the 'up' state but during the transition from the 'up' to the 'down' state of the slow wave, a period of high synchronization. Epileptic discharges appear therefore more associated with synchronization than with excitability. Furthermore, high frequency oscillations in channels devoid of epileptic activity peak differently during the slow wave cycle from those in channels with epileptic activity. This property may allow differentiating physiological from pathological high frequency oscillations, a problem that is unresolved until now

Two Seizure-Onset Types Reveal Specific Patterns of High-Frequency Oscillations in a Model of Temporal Lobe Epilepsy

High-frequency oscillations (HFOs; 80-500 Hz) are thought to mirror the pathophysiological changes occurring in epileptic brains. However, the distribution of HFOs during seizures remains undefined. Here, we recorded from the hippocampal CA3 subfield, subiculum, entorhinal cortex, and dentate gyrus to quantify the occurrence of ripples (80-200 Hz) and fast ripples (250-500 Hz) during low-voltage fast-onset (LVF) and hypersynchronous-onset (HYP) seizures in the rat pilocarpine model of temporal lobe epilepsy. We discovered in LVF seizures that (1) progression from preictal to ictal activity was characterized in seizure-onset zones by an increase of ripple rates that were higher when compared with fast ripple rates and (2) ripple rates during the ictal period were higher compared with fast ripple rates in seizure-onset zones and later in regions of secondary spread. In contrast, we found in HYP seizures that (1) fast ripple rates increased during the preictal period and were higher compared with ripple rates in both seizure-onset zones and in regions of secondary spread and (2) they were still higher compared with ripple rates in both seizure-onset zones and regions of secondary spread during the ictal period. Our findings demonstrate that ripples and fast ripples show distinct time- and region-specific patterns during LVF and HYP seizures, thus suggesting that they play specific roles in ictogenesis. © 2012 the authors