Martin Vreugdenhil

Anticonvulsant effect of polyunsaturated fatty acids in rats, using the cortical stimulation model

Recent studies have shown that long-chain polyunsaturated fatty acids can prevent cardiac arrhythmias, attributed to the reduction in excitability of cardiomyocytes, owing mainly to a shift in hyperpolarizing direction of the inactivation curves of both Na+ and Ca2+ currents and to a slowed recovery from inactivation. Qualitatively similar effects of polyunsaturated fatty acids on inactivation parameters have been observed in freshly isolated hippocampal neurons. Since the same effects are presumed to underlie the action of some established anticonvulsant drugs, polyunsaturated fatty acids might have an anticonvulsant action as well. We have investigated this for eicosapentaenoic acid, docosahexaenoic acid, linoleic acid and oleic acid, employing cortical stimulation in rats, a seizure model allowing the determination of the full anticonvulsant effect-time profile in freely moving, individual animals. I.v. infusion of 40 micromol of eicosapentaenoic acid or docosahexaenoic acid over a period of 30 min, modestly increased the threshold for localized seizure activity after 6 h by 73 +/- 13 microA (mean +/- S.E.M.; n = 7) and 77 +/- 17 microA (n = 7), respectively, and the threshold for generalized seizure activity by 125 +/- 20 and 130 +/- 19 microA, respectively (P < 0.001). The thresholds remained elevated for 6 h after infusion, but returned to baseline the next day. Free plasma concentrations in rats treated with eicosapentaenoic acid or docosahexaenoic acid, averaged 5.7 +/- 1.6 microM (n = 4) for eicosapentaenoic acid and 12.9 +/- 1.8 microM (n = 5) for docosahexaenoic acid at the end of infusion, but declined to undetectable levels within 3 h. Linoleic acid and oleic acid were less effective. Possible mechanisms for the modest anticonvulsant effect but of long duration with the polyunsaturated fatty acids are discussed.

Parvalbumin deficiency affects network properties resulting in increased susceptibility to epileptic seizures

Networks of GABAergic interneurons are of utmost importance in generating and promoting synchronous activity and are involved in producing coherent oscillations. These neurons are characterized by their fast-spiking rate and by the expression of the Ca(2+)-binding protein parvalbumin (PV). Alteration of their inhibitory activity has been proposed as a major mechanism leading to epileptic seizures and thus the role of PV in maintaining the stability of neuronal networks was assessed in knockout (PV-/-) mice. Pentylenetetrazole induced generalized tonic-clonic seizures in all genotypes, but the severity of seizures was significantly greater in PV-/- than in PV+/+ animals. Extracellular single-unit activity recorded from over 1000 neurons in vivo in the temporal cortex revealed an increase of units firing regularly and a decrease of cells firing in bursts. In the hippocampus, PV deficiency facilitated the GABA(A)ergic current reversal induced by high-frequency stimulation, a mechanism implied in the generation of epileptic activity. We postulate that PV plays a key role in the regulation of local inhibitory effects exerted by GABAergic interneurons on pyramidal neurons. Through an increase in inhibition, the absence of PV facilitates synchronous activity in the cortex and facilitates hypersynchrony through the depolarizing action of GABA in the hippocampus.

High-Frequency Network Activity, Global Increase in Neuronal Activity, and Synchrony Expansion Precede Epileptic Seizures In Vitro

How seizures start is a major question in epilepsy research. Preictal EEG changes occur in both human patients and animal models, but their underlying mechanisms and relationship with seizure initiation remain unknown. Here we demonstrate the existence, in the hippocampal CA1 region, of a preictal state characterized by the progressive and global increase in neuronal activity associated with a widespread buildup of low-amplitude high-frequency activity (HFA) (>100 Hz) and reduction in system complexity. HFA is generated by the firing of neurons, mainly pyramidal cells, at much lower frequencies. Individual cycles of HFA are generated by the near-synchronous (within ∼5 ms) firing of small numbers of pyramidal cells. The presence of HFA in the low-calcium model implicates nonsynaptic synchronization; the presence of very similar HFA in the high-potassium model shows that it does not depend on an absence of synaptic transmission. Immediately before seizure onset, CA1 is in a state of high sensitivity in which weak depolarizing or synchronizing perturbations can trigger seizures. Transition to seizure is characterized by a rapid expansion and fusion of the neuronal populations responsible for HFA, associated with a progressive slowing of HFA, leading to a single, massive, hypersynchronous cluster generating the high-amplitude low-frequency activity of the seizure.

CALCIUM CURRENTS IN PYRAMIDAL CA1 NEURONS IN VITRO AFTER KINDLING EPILEPTOGENESIS IN THE HIPPOCAMPUS OF THE RAT

Calcium is an important second messenger which plays a role in the regulation of neuronal excitability and in many forms of synaptic plasticity. In kindling epileptogenesis, a model of focal epilepsy, calcium plays an important role. The in situ patch-clamp technique was used to record calcium currents in slices obtained from kindled rats and controls. We found that low-voltage-activated calcium currents, probably of dendritic origin, were larger after kindling (80%). The transient high-voltage-activated calcium currents were also enhanced after kindling (50% higher). The increase of the current is accompanied by a decrease in the time constant of inactivation. The change was still present six weeks after the kindling stimulations were stopped. These data demonstrate that low-voltage-activated calcium currents are involved in epileptogenesis. Their enhancement in the dendrites will boost synaptic depolarization and result in enhanced calcium influx, which is critically dependent on the specific activation pattern.

Modulation of sodium currents in rat CA1 neurons by carbamazepine and valproate after kindling epileptogenesis

Summary: Purpose: To determine the modulation of sodium currents in hippocampal CA1 neurons by carbamazepine (CBZ) and valproate (VPA), before and after kindling epileptogenesis.

ERK activation causes epilepsy by stimulating NMDA receptor activity

The ERK MAPK signalling pathway is a highly conserved kinase cascade linking transmembrane receptors to downstream effector mechanisms. To investigate the function of ERK in neurons, a constitutively active form of MEK1 (caMEK1) was conditionally expressed in the murine brain, which resulted in ERK activation and caused spontaneous epileptic seizures. ERK activation stimulated phosphorylation of eukaryotic translation initiation factor 4E (eIF4E) and augmented NMDA receptor 2B (NR2B) protein levels. Pharmacological inhibition of NR2B function impaired synaptic facilitation in area cornus ammonicus region 3 (CA3) in acute hippocampal slices derived from caMEK1‐expressing mice and abrogated epilepsy in vivo. In addition, expression of caMEK1 caused phosphorylation of the transcription factor, cAMP response element‐binding protein (CREB) and increased transcription of ephrinB2. EphrinB2 overexpression resulted in increased NR2B tyrosine phosphorylation, which was essential for caMEK1‐induced epilepsy in vivo, since conditional inactivation of ephrinB2 greatly reduced seizure frequency in caMEK1 transgenic mice. Therefore, our study identifies a mechanism of epileptogenesis that links MAP kinase to Eph/Ephrin and NMDA receptor signalling.

Enhancement of calcium currents in rat hippocampal CA1 neurons induced by kindling epileptogenesis

Kindling of the Schaffer collaterals in the dorsal hippocampus of the rat induced an epileptogenic focus in area CA1. Pyramidal neurons were acutely isolated from this area in fully kindled rats one day after the last class five generalized seizure. Calcium currents were measured in these cells under the whole-cell patch voltage-clamp condition after blockade of sodium and potassium currents. Voltage-dependent calcium currents were activated by depolarizing voltage steps from different prepulse potentials. Calcium currents activated at 0 mV consisted of a sustained component and two voltage-dependent inactivating components. Current inactivation was fitted with two exponentials (time-constants of 13 and 72 ms) and a constant. When cells from kindled rats were compared with those from controls, the amplitudes of the slow-inactivating and the sustained component were significantly enhanced by 36% and 39%, respectively; the fast inactivating current showed only a small enhancement. Inactivation kinetics, time-to-peak and voltage dependency of activation and steady-state inactivation were unchanged. Shape and size of the analysed cells from kindled rats were not different from those in controls. We concluded that an increased specific calcium conductance of as yet unknown origin underlies the larger current. The magnitude of the observed changes is such that it will considerably increase calcium influx and consequently raise intracellular calcium concentration during tetanic stimulation and subsequent periods of paroxysmal activity. This increase will modulate calcium-dependent factors that regulate neuronal excitability and may lead to the enhanced excitability found in kindled tissue.

Persistent sodium current in subicular neurons isolated from patients with temporal lobe epilepsy

The persistent sodium current is a common target of anti‐epileptic drugs and contributes to burst firing. Intrinsically burst firing subicular neurons are involved in the generation and spread of epileptic activity. We measured whole‐cell sodium currents in pyramidal neurons isolated from the subiculum resected in drug‐resistant epileptic patients and in rats. In half of the cells from both patients and rats, the sodium current inactivated within 500 ms at −30 mV. Others displayed a tetrodotoxin‐sensitive slowly or non‐inactivating sodium current of up to 53% of the total sodium current amplitude. Compared with the transient sodium current in the same cells, this persistent sodium current activated with normal kinetics but its voltage‐dependent activation occurred 7 mV more hyperpolarized. Depolarizing voltage steps that lasted 10 s completely inactivated the persistent sodium current. Its voltage dependence did not differ from that of the transient sodium current but its slope was less steep. The voltage dependence and kinetics of the persistent sodium current in cells from patients were not different from that in subicular cells from rats. The current density and the relative amplitude contribution were 3–4 times greater in neurons from drug‐resistant epilepsy patients. The abundant presence of persistent sodium current in half of the subicular neurons could lead to a larger number of neurons with intrinsic burst firing. The extraordinarily large amplitude of the persistent sodium current in this subset of subicular neurons might explain why these patients are susceptible to seizures and hard to treat pharmacologically.

Kindling-induced long-lasting enhancement of calcium current in hippocampal CA1 area of the rat: Relation to calcium-dependent inactivation

Daily tetanization of the Schaffer collaterals (kindling) in the rat hippocampus induces a persistent epileptogenic focus in area CA1. Neurons were enzymatically isolated from the focal region one day or six weeks after seven class V generalized seizures had been evoked. Calcium currents were measured under voltage-clamp conditions in the whole-cell patch configuration. One day after kindling, as well as six weeks later, the amplitudes of a slow-inactivating (tau = 90 ms) and a non-inactivating calcium current component were, in comparison to controls, enhanced by 30 and 40%, respectively. This enhancement was therefore related to the kindled state of enhanced excitability. The enhancement of the calcium current was independent of the steady-state intracellular calcium concentration. Fast calcium-dependent inactivation was provoked with double-pulse protocols that conditioned the neuron with a defined calcium-influx in the first pulse. Despite the larger calcium current during the conditioning pulse, the relative calcium-dependent inactivation of the sustained current component was reduced in neurons from the kindled focus. Repetitive depolarizations, once every second, evoked a cumulative calcium-dependent inactivation. Nothwithstanding the larger calcium current, kindling also persistently reduced this slow inactivation of both transient and sustained high threshold calcium current. The reduction in calcium-dependent inactivation cannot be responsible for the increased current, but can certainly enhance the calcium influx during prolonged activation or seizures. The changes can be explained by assuming that additional calcium channels are recruited at a location that prevents calcium-dependent inactivation.

Effect of valproic acid on sodium currents in cortical neurons from patients with pharmaco-resistant temporal lobe epilepsy

In a selected group of temporal lobe epilepsy patients with seizures refractory to pharmacological treatment, pharmacological seizure control can be attained by surgical resection of the epileptic zone. We investigated to what extent pharmaco-resistance is reflected in a reduced response at the cellular level, in neurons acutely isolated from the temporal cortex resected in 20 patients. We studied the effect of valproic acid (VPA) on the transient sodium current, measured under whole-cell voltage-clamp conditions. We compared neurons from patients with temporal lobe sclerosis (S) with neurons from patients without hippocampal sclerosis (nS) and compared hippocampal CA1 neurons (CA) with neocortical neurons (NC). We could not detect differences in the voltage dependence and kinetics of sodium current activation and inactivation in any of the group comparisons. VPA shifted the voltage dependence of steady-state inactivation (expressed as V(h,i) in a Boltzmann fit) to more hyperpolarized levels. The shift induced by 2 mM VPA was -5.1 +/- 0.7 mV in CA-S (n = 13), -5.1 +/- 0.7 mV in CA-nS (n = 25), -4.3 +/- 0.5 mV in NC-S (n = 17) and -4.9 +/- 0.5 mV in NC-nS (n = 16) The relation between concentration and voltage shift had an EC50 of 1.4 +/- 0.2 mM VPA (n = 16) and a maximal shift of 9.6 +/- 0.9 mV. We conclude that pharmaco-resistance in these patients is not associated with a changed modulation of the sodium current by VPA. Results are discussed in the light of a reduced sodium current modulation by carbamazepine in CA1 neurons of patients with hippocampal sclerosis and of similar observations in the kindling model of epileptogenesis.