Pathophysiogenesis of mesial temporal lobe epilepsy: is prevention of damage antiepileptogenic?

Temporal lobe epilepsy (TLE) is frequently associated with hippocampal sclerosis, possibly caused by a primary brain injury that occurred a long time before the appearance of neurological symptoms. This type of epilepsy is characterized by refractoriness to drug treatment, so to require surgical resection of mesial temporal regions involved in seizure onset. Even this last therapeutic approach may fail in giving relief to patients. Although prevention of hippocampal damage and epileptogenesis after a primary event could be a key innovative approach to TLE, the lack of clear data on the pathophysiological mechanisms leading to TLE does not allow any rational therapy. Here we address the current knowledge on mechanisms supposed to be involved in epileptogenesis, as well as on the possible innovative treatments that may lead to a preventive approach. Besides loss of principal neurons and of specific interneurons, network rearrangement caused by axonal sprouting and neurogenesis are well known phenomena that are integrated by changes in receptor and channel functioning and modifications in other cellular components. In particular, a growing body of evidence from the study of animal models suggests that disruption of vascular and astrocytic components of the blood-brain barrier takes place in injured brain regions such as the hippocampus and piriform cortex. These events may be counteracted by drugs able to prevent damage to the vascular component, as in the case of the growth hormone secretagogue ghrelin and its analogues. A thoroughly investigation on these new pharmacological tools may lead to design effective preventive therapies.

Neurosteroids and epileptogenesis.

Epileptogenesis is defined as the latent period at the end of which spontaneous recurrent seizures occur. This concept has been recently re-evaluated to include exacerbation of clinically-manifested epilepsy. Thus, in patients affected by pharmacoresistant seizures, the progression toward a worse condition may be viewed as the result of a durable epileptogenic process. However, the mechanism potentially responsible for this progression remains unclear. Neuroinflammation has been consistently detected both in the latent period and in the chronic phase of epilepsy, especially when brain damage is present. This phenomenon is accompanied by glial cell reaction, leading to gliosis. We have previously described rats presenting an increased expression of the cytochrome P450 cholesterol side-chain cleavage (P450scc) enzyme, during the latent period, in glial cells of the hippocampus. The P450scc enzyme is critically involved in the synthesis of neurosteroids and its up-regulation is associated with a delayed appearance of spontaneous recurrent seizures in rats that experienced status epilepticus induced by pilocarpine. Moreover, by decreasing the synthesis of neurosteroids able to promote inhibition, such as allopregnanolone, through the administration of the 5α-reductase blocker finasteride, it is possible to terminate the latent period in pilocarpine-treated rats. Finasteride was also found to promote seizures in the chronic period of epileptic rats, suggesting that neurosteroids are continuously produced to counteract seizures. In humans, exacerbation of epilepsy has been also described in patients occasionally exposed to finasteride. Overall, these findings suggest a major role of neurosteroids in the progression of epilepsy and a possible antiepileptogenic role of allopregnanolone and cognate molecules.

Increased perivascular laminin predicts damage to astrocytes in CA3 and piriform cortex following chemoconvulsive treatments.

Status epilepticus (SE) induced by pilocarpine or kainate is associated with yet not systemically investigated astrocytic and vascular injuries. To investigate their possible association with neuronal damage, the changes in glial fibrillary acidic protein (GFAP), laminin and neuron-specific nuclear protein (NeuN) immunoreactivities were analyzed in rats treated with pilocarpine (380 mg/kg) or kainate (15 mg/kg), and receiving diazepam (20mg/kg) after 10 min of SE. A different group of rats was injected with endothelin-1 (ET-1) in the caudate putamen to reproduce the changes in GFAP and laminin immunoreactivities associated with ischemia. Focal loss of GFAP immunostaining was accompanied by increased laminin immunoreactivity in blood vessels, in all the examined groups. Regression analysis revealed a significant (P<0.01) relationship between astrocytic lesion and increased laminin immunoreactivity in the piriform cortex (Pir) of both pilocarpine (R(2)=0.88) and kainate (R(2)=0.94) groups of treatment. A significant relationship (P<0.01; R(2)=0.81) was also present in the cornu Ammonis 3 (CA3) hippocampal region of pilocarpine-treated rats. At variance, neuronal and glial lesions were significantly related (P<0.05, R(2)=0.74) only in the substantia nigra of pilocarpine-treated rats. The ratio between areas of GFAP and laminin changes of immunoreactivity in the ET-1 group was similar to those found in pilocarpine- and kainate-treated rats in specific brain regions, such as the hippocampal CA3 subfield, Pir and the anterior olfactory nucleus. The amygdala and submedius thalamic nucleus in the pilocarpine group, and the perirhinal and entorhinal cortices in the kainate group, also presented ischemic-like changes. These results indicate that laminin immunoreactivity is upregulated in the basal lamina of blood vessels after SE induced by pilocarpine or kainate. This phenomenon is significantly associated with lesions involving more glial than neuronal cells, in specific cerebral regions.

Zn(2+) slows down Ca(V)3.3 gating kinetics: implications for thalamocortical activity.

We employed whole cell patch-clamp recordings to establish the effect of Zn(2+) on the gating the brain specific, T-type channel isoform Ca(V)3.3 expressed in HEK-293 cells. Zn(2+) (300 microM) modified the gating kinetics of this channel without influencing its steady-state properties. When inward Ca(2+) currents were elicited by step depolarizations at voltages above the threshold for channel opening, current inactivation was significantly slowed down while current activation was moderately affected. In addition, Zn(2+) slowed down channel deactivation but channel recovery from inactivation was only modestly changed. Zn(2+) also decreased whole cell Ca(2+) permeability to 45% of control values. In the presence of Zn(2+), Ca(2+) currents evoked by mock action potentials were more persistent than in its absence. Furthermore, computer simulation of action potential generation in thalamic reticular cells performed to model the gating effect of Zn(2+) on T-type channels (while leaving the kinetic parameters of voltage-gated Na(+) and K(+) unchanged) revealed that Zn(2+) increased the frequency and the duration of burst firing, which is known to depend on T-type channel activity. In line with this finding, we discovered that chelation of endogenous Zn(2+) decreased the frequency of occurrence of ictal-like epileptiform discharges in rat thalamocortical slices perfused with medium containing the convulsant 4-aminopyridine (50 microM). These data demonstrate that Zn(2+) modulates Ca(V)3.3 channel gating thus leading to increased neuronal excitability. We also propose that endogenous Zn(2+) may have a role in controlling thalamocortical oscillations.

Antiepileptic drugs and muscarinic receptor-dependent excitation in the rat subiculum.

Field and intracellular recordings were made in an in vitro slice preparation to establish whether the antiepileptic drugs topiramate and lamotrigine modulate cholinergic excitation in the rat subiculum. Bath application of carbachol (CCh, 70-100microM) induced: (i) spontaneous and synchronous field oscillations (duration=up to 7s) that were mirrored by intracellular depolarizations with rhythmic action potential bursts; and (ii) depolarizing plateau potentials (DPPs, duration=up to 2.5s) associated with action potential discharge in response to brief (50-100ms) intracellular depolarizing current pulses. Ionotropic glutamatergic receptor antagonists abolished the field oscillations without influencing DPPs, while atropine (1microM) markedly reduced both types of activity. Topiramate (10-100microM, n=8-13 slices) or lamotrigine (50-400microM, n=3-12) decreased in a dose-dependent manner, and eventually abolished, CCh-induced field oscillations. During topiramate application, these effects were accompanied by marked DPP reduction. When these antiepileptic drugs were tested on DPPs recorded in the presence of CCh+ionotropic glutamatergic and GABA receptor antagonists, only topiramate reduced DPPs (n=5-19/dose; IC(50)=18microM, n=48). Similar effects were induced by topiramate during metabotropic glutamate receptor antagonism (n=5), which did not influence DPPs. Thus, topiramate and lamotrigine reduce CCh-induced epileptiform synchronization in the rat subiculum but only topiramate is effective in controlling DPPs. We propose that muscarinic receptor-mediated excitation represents a target for the action of some antiepileptic drugs such as topiramate.

Phosphorylation of sodium channels mediated by protein kinase-C modulates inhibition by topiramate of tetrodotoxin-sensitive transient sodium current.

BACKGROUND AND PURPOSE: Topiramate is a novel anticonvulsant known to modulate the activity of several ligand- and voltage-gated ion channels in neurons. The mechanism of action of topiramate, at a molecular level, is still unclear, but the phosphorylation state of the channel/receptor seems to be a factor that is able to influence its activity. We investigated the consequences of phosphorylation of the sodium channel on the effect of topiramate on tetrodotoxin (TTX)-sensitive transient Na(+) current (I(NaT)). EXPERIMENTAL APPROACH: I(NaT) was recorded in dissociated neurons of rat sensorimotor cortex using whole-cell patch-clamp configuration. KEY RESULTS: We found that topiramate (100 microM) significantly shifted the steady-state I(NaT) inactivation curve in a hyperpolarized direction. In neurons pre-treated with a PKC-activator, 1-oleoyl-2-acetyl-sn-glycerol (OAG; 2 microM), the net effect of topiramate on steady-state I(NaT) inactivation was significantly decreased. In addition, OAG also slightly shifted the I(NaT) activation curve in a hyperpolarized direction, while perfusion with topiramate had no effect on the parameters of I(NaT) activation. CONCLUSIONS AND IMPLICATIONS: These data show that PKC-activation can modulate the effect of topiramate on I(NaT). This suggests that channel phosphorylation in physiological or pathological conditions (such as epiliepsy), can alter the action of topiramate on sodium currents.

Layer-specific properties of the persistent sodium current in sensorimotor cortex.

We evaluated the characteristics of the persistent sodium current (I(NaP)) in pyramidal neurons of layers II/III and V in slices of rat sensorimotor cortex using whole cell patch-clamp recordings. In both layers, I(NaP) began activating around -60 mV and was half-activated at -43 mV. The I(NaP) peak amplitude and density were significantly higher in layer V. The voltage-dependent I(NaP) steady-state inactivation occurred at potentials that were significantly more positive in layer V (V(1/2): -42.3 +/- 1.1 mV) than in layer II/III (V(1/2): -46.8 +/- 1.6 mV). In both layers, a current fraction corresponding to about 25% of the maximal peak amplitude did not inactivate. The time course of I(NaP) inactivation and recovery from inactivation could be fitted with a biexponential function. In layer V pyramidal neurons the faster time constant of development of inactivation had variable values, ranging from 158.0 to 1,133.8 ms, but it was on average significantly slower than that in layer II/III (425.9 +/- 80.5 vs. 145.8 +/- 18.2 ms). In both layers, I(NaP) did not completely inactivate even with very long conditioning depolarizations (40 s at -10 mV). Recovery from inactivation was similar in the two layers. Layer V intrinsically bursting and regular spiking nonadapting neurons showed particularly prolonged depolarized plateau potentials when Ca2+ and K+ currents were blocked and slower early phase of I(NaP) development of inactivation. The biexponential kinetics characterizing the time-dependent inactivation of I(NaP) in layers II/III and V indicates a complex inactivating process that is incomplete, allowing a residual "persistent" current fraction that does not inactivate. Moreover, our data indicate that I(NaP) has uneven inactivation properties in pyramidal neurons of different layers of rat sensorimotor cortex. The higher current density, the rightward shifted voltage dependency of inactivation as well the slower kinetics of inactivation characterizing I(NaP) in layer V with respect to layer II/III pyramidal neurons may play a significant role in their ability to fire recurrent action potential bursts, as well in the high susceptibility to generate epileptic events.

Protein-kinase C-dependent phosphorylation inhibits the effect of the antiepileptic drug topiramate on the persistent fraction of sodium currents.

We investigated the interference of protein-kinase C (PKC)-dependent Na(+) channel phosphorylation on the inhibitory effect that the antiepileptic drug topiramate (TPM) has on persistent Na(+) currents (I(NaP)) by making whole cell patch-clamp and intracellular recordings of rat sensorimotor cortex neurons. The voltage-dependent activation of I(NaP) was significantly shifted in the hyperpolarizing direction when PKC was activated by 1-oleoyl-2-acetyl-sn-glycerol (OAG). TPM reduced the peak amplitude of I(NaP), but it did not counteract the OAG-induced shift in I(NaP) activation. Firing property experiments showed that the firing threshold was lowered by OAG. TPM was unable to counteract this effect, which may be due to OAG-dependent enhancement of the contribution of subthreshold I(NaP). These data suggest that PKC activation may limit the effect of the anticonvulsant TPM on the persistent fraction of Na(+) currents. The channel phosphorylation that may occur in cortical neurons as a result of physiological or pathological (e.g. epileptic) events can modulate the action of TPM on Na(+) currents.

Resveratrol derivatives and their role as potassium channels modulators.

A series of stilbenoid analogues of resveratrol (trans-3,4',5-trihydroxystilbene) with a stilbenic or a bibenzylic skeleton have been prepared by partial synthesis from resveratrol and dihydroresveratrol. The synthesized compounds have been evaluated for their ability to modulate voltage-gated channels.