Cristina Zona

Topiramate attenuates voltage-gated sodium currents in rat cerebellar granule cells

Whole-cell, voltage-clamp recordings were made from rat cerebellar granule cells in culture under experimental conditions designed to study voltage-gated Na+ currents that were elicited by depolarizing commands from a holding potential of -60 mV up to +20 mV. These tetrodotoxin-sensitive inward currents were reduced in a dose-related manner by bath application of the structurally novel, anticonvulsant drug topiramate (10-1000 microM; n = 16). Dose-response analysis of this effect revealed an IC50 of 48.9 microM. Topiramate also made the steady-state inactivation curve of this current shift toward more negative values (midpoint of the inactivation curve -46.9 mV under control conditions and -56.5 mV during topiramate application; n = 5). We propose that these effects may contribute to control the sustained depolarizations with repetitive firing of action potentials that occur within neuronal networks during seizure activity. Therefore they may represent a mechanism of action for this novel anticonvulsant drug.

Levetiracetam does not modulate neuronal voltage-gated Na+and T-type Ca2+currents

This study investigated whether the mechanism of action of levetiracetam (LEV) is related to effects on neuronal voltage-gated Na+ or T-type Ca2+currents. Rat neocortical neurones in culture were subjected to the whole-cell mode of voltage clamping under experimental conditions designed to study voltage-gated Na+ current. Additionally, visually identified pyramidal neurones in the CA1 area of rat hippocampal slices were subjected to the whole-cell mode of voltage clamping under experimental conditions designed to study low-voltage-gated (T-type) Ca2+ current. LEV (10 microM-1 mM) did not modify the Na+ current amplitude and did not change (200 microM) the steady-state activation and inactivation, the time to peak, the fast kinetics of the inactivation and the recovery from the steady-state inactivation of the Na+ current. Likewise, LEV (32-100 microM) did not modify the amplitude and did not change the steady-state activation and inactivation, the time to peak, the fast kinetics of the inactivation and the recovery from the steady-state inactivation of the T-type Ca2+current. In conclusion, neuronal voltage-gated Na+ channels do not appear directly involved in the antiepileptic mechanism of action of LEV, and LEV was devoid of effect on the low-voltage-gated (T-type) Ca2+ current in hippocampal neurones.

Interleukin-2 suppresses established long-term potentiation and inhibits its induction in the rat hippocampus

The effects of recombinant interleukin-2 (rIL-2) on the potentiation of the synaptic transmission were studied in rat hippocampal slices by using extracellular field potential recordings. The application of rIL-2 inhibited the induction of both short-term (STP) and long-term potentiation (LTP) in a dose-dependent manner. In addition, rIL-2 (1000 U/ml) reduced both post-tetanic potentiation (PTP) and LTP maintenance phase. The possible involvement of rIL-2 action on the synaptic potentiation with the enzymatic activity of protein kinase systems is discussed.

Low magnesium epileptogenesis in the rat hippocampal slice: electrophysiological and pharmacological features

Extra- and intracellular recording techniques were used to study the epileptiform activity generated by rat hippocampal slices perfused with Mg2(+)-free artificial cerebrospinal fluid (ACSF). This procedure induced in both CA1 and CA3 subfields the appearance of synchronous, spontaneously occurring epileptiform discharges which consisted of extracellularly recorded 100-800 ms long, positive shifts with superimposed negative going population spikes. Simultaneous, extracellular recordings from CA1 and CA3 subfields revealed that the epileptiform discharges in CA3 preceded those occurring in CA1 by 5-25 ms. Surgical separation of the two areas led to the disappearance of spontaneous events in the CA1 but not in the CA3 subfield. In this type of experiment CA1 pyramidal cells still generated epileptiform discharges following orthodromic stimuli. The intracellular counterpart of both spontaneous and stimulus-induced epileptiform discharges in CA1 and CA3 pyramidal cells was a large amplitude depolarization with high frequency discharge of action potentials which closely resembled the paroxysmal depolarizing shift recorded in the experimental epileptogenic focus. A hyperpolarizing potential triggered by alvear stimuli was recorded in CA1 cells perfused with Mg2(+)-free ACSF. This hyperpolarization was blocked by bicuculline methiodide (BMI) indicating that it represented a GABAergic inhibitory postsynaptic potential (IPSP). BMI also caused a prolongation of both spontaneous and stimulus-induced Mg(+)-free epileptiform discharges. Perfusion of the slices with the N-methyl-D-aspartate (NMDA) receptor antagonist DL-2-amino-5-phosphono-valerate (APV) reduced and eventually abolished the Mg(+)-free epileptiform discharges. These effects were more pronounced in the CA1 than in the CA3 subfield. APV also reduced the amplitude and the duration of the alveus-induced IPSP. These data demonstrate that Mg(+)-free epileptiform activity is present in the hippocampal slice at a time when inhibitory GABAergic potentials are operant as well as that in the CA1 subfield this type of epileptiform activity is dependent upon NMDA-activated conductances. Our experiments also indicate that NMDA receptors might be involved in the neuronal circuit responsible for the hyperpolarizing IPSP generated by CA1 pyramidal neurons.

Riluzole interacts with voltage-activated sodium and potassium currents in cultured rat cortical neurons

The actions of the neuroprotective and anticonvulsant agent riluzole on voltage-activated currents were studied in primary cultures of rat cortical neurons by using whole-cell patch-clamp recording techniques. Isolated Na+, Ca2+ and K+ currents were generated in these cells by depolarizing commands from a holding potential of - 80 mV. Riluzole (10-300 microM) reversibly reduced in a dose-dependent manner the inward Na+ currents with an IC50 of 51 microM in all the tested neurons (n=29). This drug also shifted the steady-state inactivation curve of the sodium current towards more negative values (about 20mV, n=15) while it did not change significantly the decay phase of the Na+ current. Furthermore, riluzole (100 and 300 microM; n=5 and n=3, respectively) did not modulate the inward Ca2+ currents evoked by depolarizing steps on cortical cells. An additional concentration-dependent effect of riluzole was observed on the outward potassium currents. In fact, while the amplitude of the peak of the outward current (IA) was not changed significantly, the amplitude of the late component of the outward K+ current (Iss) was markedly decreased during the perfusion of riluzole (IC50=88 microM; n=16). It is concluded that riluzole modulates the Na+- and the late K+-dependent currents in cortical neurons. Both phenomena may explain, at least in part, the anticonvulsant and neuroprotective properties of this compound.

Enhancement of learning and memory after activation of cerebral Rho GTPases.

The mechanism whereby the morphology and connectivity of the dendritic tree is regulated depends on an actin dynamics that, in turn, is controlled by Rho GTPases, a family of small GTP-binding proteins encompassing Rho, Rac, and Cdc42 subfamilies. Cytotoxic necrotizing factor 1 (CNF1), a protein toxin from Escherichia coli, constitutively activates Rho GTPases, thus leading to remodeling of the actin cytoskeleton in intact cells. Here, we show that the modulation of cerebral RhoA and Rac1 activity induced by CNF1 in mice leads to (i) rearrangement of cerebral actin cytoskeleton, (ii) enhanced neurotransmission and synaptic plasticity, and (iii) improved learning and memory in various behavioral tasks. The effects persist for weeks and are not observed in mice treated with a recombinant CNF1, in which the enzymatic activity was abolished by substituting serine to cysteine at position 866. The results suggest that learning ability can be improved through pharmacological manipulation of neural connectivity.

Altered excitability of motor neurons in a transgenic mouse model of familial amyotrophic lateral sclerosis

Various evidence suggests that amyotrophic lateral sclerosis (ALS) selectively affects motor neuron functioning, but electrophysiological alterations of single motor neurons in ALS remains to be documented. In the present work, the excitability of motor neurons has been tested in a transgenic mouse model of a familial form of ALS, associated with a mutation in Cu,Zn superoxide dismutase (Gly(93)-->Ala). Patch-clamp recordings of membrane potential in transgenic mice motor neurons showed that they fire with increased frequency and shorter duration compared to motor neurons from control mice. The passive membrane properties of these neurons were equivalent however. Such results suggest that an altered motor neuron excitability accompanies an ALS associated mutation and that may contribute to the pathogenesis of the disease.

Increased persistent sodium current determines cortical hyperexcitability in a genetic model of amyotrophic lateral sclerosis

Cortical hyperexcitability has been observed in Amyotrophic Lateral Sclerosis (ALS) patients. Familial ALS accounts for 10% of all cases and mutations of the Cu,Zn superoxide dismutase (SOD1) gene have been identified in about 20% of the familial cases. The aim of this study was to investigate whether in a mouse model of ALS the cortical neurons developed hyperexcitability due to intrinsic properties of the single cell. We first examined the passive membrane properties and the pattern of repetitive firing in cultured cortical neurons from Control mice and transgenic mice expressing high levels of the human mutated protein (Gly(93)-->Ala, G93A). The former did not display significantly differing values between Control and G93A cortical neurons. However, the threshold potential and time of the first action potential decreased significantly and the firing frequency increased significantly in the G93A compared to Control neurons. The analysis of the voltage-dependent sodium currents revealed that the fast transient sodium current was unaffected by the SOD1 mutation whereas the persistent sodium current was significantly higher in the mutated neurons. Finally, Riluzole, a selective blocker of the persistent sodium current at low concentrations, decreased the firing frequency in G93A neurons, strongly indicating an involvement of this current in the observed hyperexcitability. These are the first data that demonstrate an intrinsic hyperexcitability in the G93A cortical neurons due to a higher current density of the persistent sodium current in the mutated neurons and open up new prospects of understanding ALS disease etiopathology.

Cu/Zn-superoxide dismutase (GLY93-->ALA) mutation alters AMPA receptor subunit expression and function and potentiates kainate-mediated toxicity in motor neurons in culture.

The cause of the selective degeneration of motor neurons in amyotrophic lateral sclerosis (ALS) remains a mystery. One potential pathogenic mechanism is excitotoxicity due to disturbances of glutamatergic neurotransmission, particularly via AMPA-sensitive glutamate receptors. We report here that motor neurons from a familial ALS-linked superoxide dismutase (SOD1) mutant G93A mouse show an higher susceptibility to kainate-induced excitotoxicity. Moreover, they expressed GluR(3) and GluR(4) mRNA at detectable levels more frequently, with a modified electrophysiology when compared with control and wild-type SOD1 motor neurons. Thus, the SOD1 G93A mutation causes changes in the AMPA-receptor expression and function, as well as a susceptibility to kainate-mediated excitotoxicity, which may promote the motor neuron degeneration seen in ALS.

Lamotrigine Reduces Voltage-Gated Sodium Currents in Rat Central Neurons in Culture

Summary: Purpose: To study the mechanism or mechanisms of action of lamotrigine (LTG) and, in particular, to establish its effects on the function of NA+ channels in mammalian central neurons.