Anti-convulsive and anti-epileptic properties of brivaracetam (ucb 34714), a high-affinity ligand for the synaptic vesicle protein, SV2A.

BACKGROUND AND PURPOSE: Screening of 12,000 compounds for binding affinity to the synaptic vesicle protein 2A (SV2A), identified a high-affinity pyrrolidone derivative, brivaracetam (ucb 34714). This study examined its pharmacological profile in various in vitro and in vivo models of seizures and epilepsy, to evaluate its potential as a new antiepileptic drug. EXPERIMENTAL APPROACH: The effects of brivaracetam and levetiracetam on epileptiform activity and seizure expression were examined in rat hippocampal slices, corneally kindled mice, audiogenic seizure-susceptible mice, maximal electroshock and pentylenetetrazol seizures in mice, hippocampal-kindled rats, amygdala-kindled rats and genetic absence epilepsy rats. KEY RESULTS: Brivaracetam and levetiracetam reduced epileptiform responses in rat hippocampal slices, brivaracetam being most potent. Brivaracetam also differed from levetiracetam by its ability to protect against seizures in normal mice induced by a maximal electroshock or maximal dose of pentylenetetrazol. In corneally kindled mice and hippocampal-kindled rats, brivaracetam induced potent protection against secondarily generalized motor seizures and showed anti-kindling properties superior to levetiracetam. In amygdala-kindled rats, brivaracetam induced a significant suppression in motor-seizure severity and, contrary to levetiracetam, reduced the after-discharge at a higher dose. Audiogenic seizure-susceptible mice were protected more potently against the expression of clonic convulsions by brivaracetam than by levetiracetam. Brivaracetam induced a more complete suppression of spontaneous spike-and-wave discharges in genetic absence epilepsy rats than levetiracetam. CONCLUSIONS AND IMPLICATIONS: Brivaracetam has higher potency and efficacy than levetiracetam as an anti-seizure and anti-epileptogenic agent in various experimental models of epilepsy, and a wide therapeutic index.

Levetiracetam opposes the action of GABAA antagonists in hypothalamic neurones.

Whole-cell current-clamp recordings in guinea-pig brain slices were used to assess the effect of the novel antiepileptic drug levetiracetam (LEV; Keppra) on the gamma-aminobutyric acid-A (GABA(A)) responses evoked by exogenous applications of the agonists GABA and muscimol on hypothalamic neurones. LEV (40 microM) had no direct effect on GABA(A) responses but it occluded the GABA(A)-receptor blocking action of bicuculline-methiodide (100 microM) and, to a lesser extent, the GABA(A)-receptor blocking action of gabazine (50 microM). While previous reports have indicated that the inhibition by LEV of the epileptiform hyperexcitability induced by bicuculline in rat hippocampus might occur via non-GABAergic mechanisms, the present data suggest a possible indirect modulation by LEV of GABA-gated currents in guinea-pig hypothalamic neurones.

Can gap-junction blockade preferentially inhibit neuronal hypersynchrony vs. excitability?

Epileptic activity involves a hypersynchronous firing of large populations of brain neurons, some of which are hyperexcitable. This study explored to what extent gap-junction blockade inhibits neuronal synchronization vs. neuronal excitability. We investigated the effects of the gap-junction blockers (GJBs) 1-heptanol, 1-octanol and carbenoxolone vs. the loop diuretic furosemide on spontaneous and evoked epileptiform field potentials, induced in CA3 area of rat hippocampal slices by a 'high K(+)-low Ca(2+)' perfusion fluid. This milieu induced frequent (>30 min(-1)) spontaneous bursts, led single fimbrial stimuli to evoke repetitive population spikes (PSs), and increased PS amplitudes. Both furosemide and the three GJBs gradually reduced spontaneous field bursting, or even stopped it within one hour. The anti-bursting activity of carbenoxolone showed dose-response dependence in the concentration range 50-400 microM. 1-heptanol and 1-octanol markedly and similarly depressed all the epileptiform markers of the evoked responses, whereas carbenoxolone did not reduce the number of repetitive PSs evoked by single stimuli. By its minor effect on evoked responses, carbenoxolone resembled furosemide, rather than its congeners GJBs. These results favor the possibility that selective gap-junction blockade might antagonize epileptic synchronization, without depressing neuronal excitability.

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.

Levetiracetam inhibits the high-voltage-activated Ca(2+) current in pyramidal neurones of rat hippocampal slices.

The effect of the new antiepileptic drug levetiracetam (LEV; KEPPRA) on the neuronal high-voltage-activated (HVA) Ca(2+) current was investigated on pyramidal neurones, visually identified in the CA1 area of rat hippocampal slices. Nystatin-perforated patch clamp recordings were made under experimental conditions designed to study HVA Ca(2+) currents. The HVA current, activated by steadily increasing voltage-ramps, was reversibly eliminated by Cd(2+) and depressed by either nimodipine, or omega-Conotoxin GVIA. After 30 min perfusion of the slices with LEV 32 microM, the current decayed to 55+/-9% (mean+/-SEM; n=9) of the initial value, which is significantly (P<0.05, two-tailed t-test) lower than the rundown to 84+/-10% in a control group (n=10) of neurones. The limited, but significant depression of the neuronal HVA Ca(2+) current, produced by LEV at a clinically relevant concentration, might contribute to the antiepileptic action of the drug.

Inhibition of neuronal hypersynchrony in vitro differentiates levetiracetam from classical antiepileptic drugs.

Field potentials were recorded from rat hippocampal slices in order to compare the electrophysiological action of the new antiepileptic drug (AED), levetiracetam (LEV), with that of the classical AEDs, valproate, clonazepam and carbamazepine, on epileptiform responses induced by a 'high K(+)-low Ca(2+)' perfusion fluid. Increasing [K(+)] from 3 to 7.5 mM and decreasing [Ca(2+)] from 2.4 to 0.5 mM, in the bathing fluid, produced population spikes (PSs) of increasing amplitudes in the CA3 area of the slices, repetitive PSs evoked by single stimuli, and spontaneous bursts. Clinically relevant concentrations of LEV, 32 and 100 micro M, consistently reduced the second (PS(2)) and third (PS(3)) population spikes, and the number (N) of repetitive PSs per evoked response. Levetiracetam 32 micro M also opposed the increase in amplitude of the first PS (Delta PS(1)). Neither valproate 1 mM, nor clonazepam 1 micro M, nor carbamazepine 50 micro M, produced any decrease in Delta PS(1)and in PS(2), but all decreased N. These results show that LEV contrasts to reference AEDs by its ability to antagonize neuronal (hyper)synchronization, in the highly seizure-prone CA3 area of rat hippocampal slices.

Differential paired-pulse effects of gabazine and bicuculline in rat hippocampal CA3 area.

Field potentials were evoked in hippocampal area CA3 of anaesthetised rats by commissural stimulation, in order to study the effect of the prototypic gamma-aminobutyric acid (GABA)A antagonists gabazine (SR-95531; GBZ) and bicuculline methiodide (BMI) on paired-pulse interaction. Prominent paired-pulse inhibition of the orthodromic population spike (PS2) was observed when the interpulse interval (IPI) was < or = 40 ms, while facilitation occurred at IPIs >100 ms. Paired-pulse facilitation was lost at 500 ms. The antidromic population spike (PS1) presented paired-pulse facilitation at low-IPI, which decayed exponentially at increasing IPI. When the recording micropipettes contained millimolar concentrations of either GBZ, or BMI, single stimuli evoked repetitive (epileptiform) orthodromic PS2, of higher amplitude, while the antidromic PS1 was only weakly influenced. BMI reduced, but GBZ enhanced the low-IPI paired-pulse inhibition of the orthodromic PS2. Furthermore, BMI blunted paired-pulse facilitation of the antidromic PS1 at low-IPI, while GBZ caused strong paired-pulse inhibition of PS1 at IPI < or = 60 ms. The differential effects of GBZ and BMI on paired-pulse interaction might reflect different mechanisms of action of these compounds.

Absence of negative impact of levetiracetam on cognitive function and memory in normal and amygdala-kindled rats.

The effect of the new antiepileptic drug (AED) levetiracetam (LEV, Keppra) on cognitive function was studied in normal and amygdala-kindled rats by using the Morris water maze test. In addition, we investigated the effect of LEV on long-term potentiation (LTP) in rat hippocampal slices. Sodium valproate (VPA) was used as comparator in all studies. Clonazepam (CZP) and carbamazepine (CBZ) were used in normal rats. The results indicated that doses of LEV known to suppress motor seizures did not alter cognitive performance. In contrast, similar doses of the classic AEDs all decreased learning performance of the rats. Likewise, VPA did alter LTP but LEV was inactive. Amygdala-kindled rats were more sensitive than normal rats to the effects of VPA. These results suggest that LEV may be devoid of negative impact on cognition in epileptic patients.

Chronic electrode implantation entails epileptiform field potentials in rat hippocampal slices, similarly to amygdala kindling.

Evoked field potentials were recorded in the CA3, CA1 and dentate gyrus (DG) of hippocampal slices from amygdala kindled, non-stimulated amygdala electrode-implanted, and non-implanted age-matched rats to evaluate the consequences on hippocampal neuronal networks of kindling stimulation versus electrode implantation. No overt modification of field potentials was detected in either the CA1 or the DG areas. In contrast, a very significant increase in the occurrence of repetitive population spikes evoked by single stimuli was detected in the CA3 area in slices from both amygdala kindled and non-stimulated amygdala implanted rats. The epileptiform pattern of CA3 field potentials was at least as well expressed in implanted non-stimulated, as in kindled rats, suggesting that electrode implantation has a major contribution to this marker of epileptogenesis.

Hippocampal slices from long-term streptozotocin-injected rats are prone to epileptiform responses.

Evoked field potentials were recorded in the CA3, CA1 and dentate gyrus (DG) regions of hippocampal slices from rats injected with streptozotocin (STZ; 60 mg/kg i.p.), to detect whether STZ-induced diabetes entails changes in hippocampal excitability. No change in hippocampal responsiveness was observed in slices from diabetic rats, up to 3 weeks post-STZ. Repetitive population spikes (PSs) reminiscent of an epileptiform hyperexcitability were, however, recorded in CA3 > CA1 > DG areas after more than 4 weeks ('long-term') post-STZ, although the maximal amplitudes were not different in STZ-diabetic versus control rats. Intracellular recordings on CA3 pyramidal neurons confirmed that fimbrial stimulation evokes significantly more action potentials in neurons from 'long-term' STZ-diabetic versus control rats. This is the first report of the appearance of repetitive hippocampal responses, particularly in the seizure-prone CA3 area, as a long-term consequence of hyperglycemic STZ treatment in rat.

Thapsigargin inhibits bicuculline-induced epileptiform excitability in rat hippocampal slices.

Evoked field potentials were recorded in the CA3 region of rat hippocampal slices to detect whether intracellular Ca2+ stores are involved in the epileptiform effects of the two prototypic GABA(A) antagonists, bicuculline methiodide (BMI) and gabazine (SR-95531; GBZ). Field population spikes gradually increased and became repetitive (epileptiform bursting) in the presence of either BMI (5 microM), or GBZ (5 microM). Thapsigargin (2 microM), a depletor of intracellular Ca2+ stores, reduced the epileptiform effect of BMI, but had no significant effect on the GBZ-induced hyperexcitability. These data suggest that Ca2+ release from intracellular stores participates in the epileptiform response of hippocampal CA3 neurons to BMI, but not in the response to GBZ.

Inhibition by levetiracetam of a non-GABAA receptor-associated epileptiform effect of bicuculline in rat hippocampus.

1. Extracellular recording of field potentials, evoked by commissural stimulation in hippocampal area CA3 of anaesthetized rats, was performed in order to study the mode of action of the novel antiepileptic drug levetiracetam (ucb LO59). 2. The amplitude of orthodromic field population spike (PS2) markedly increased and repetitive population spikes appeared when the recording micropipette contained either bicuculline methiodide (BMI), or the specific GABAA antagonist gabazine (SR-95531). 3. BMI-induced increases in PS2 were reduced in a dose-dependent manner by 1 to 320 mumol kg-1 levetiracetam i.v., with a U-shape dose-response relationship. However, levetiracetam did not reduce the increases in PS2 produced by gabazine. 4. Clonazepam (1 mg kg-1, i.p.), carbamazepine (20 mg kg-1, i.p.) and valproate (200 mg kg-1, i.v.) were ineffective in preventing BMI-induced increases in PS2, while the calcium channel antagonist flunarizine, 50 mumol kg-1, i.p., reduced PS2 increments caused by BMI. The L-type calcium channel blocker nifedipine, 100 mumol kg-1, i.p., was without effect. Similar to levetiracetam, flunarizine did not reduce the increases in PS2 induced by gabazine. 5. These data suggest that the increased excitability of CA3 neurones, caused by BMI administered in situ, involves calcium-dependent processes not associated with blockade of GABAA receptors. The inhibition by levetiracetam of this calcium-dependent effect of BMI might contribute to the antiepileptic effects of the drug.

Effects of in situ manipulation of nitric oxide on rat hippocampal CA3 neurone excitability.

THE response of hippocampal CA3 neurones to commissural stimulation, as expressed by the orthodromic population spike (PS2) recorded in anaesthetized rats, was increased when the recording micropipette contained the nitric oxide (NO) synthase inhibitor N omega-nitro-L-arginine methyl ester (20 mM). The increase was stable upon repeated stimulation. When the recording micropipette contained the NO donor sodium nitroprusside (SNP; 10 mM) the amplitude of PS2 elicited by low and middle stimulus strength (Istim) rapidly decreased, while remaining unchanged at higher Istim. When the SNP-containing microelectrode was maintained in place for longer, the depression of PS2 observed at low and medium Istim disappeared and PS2 increased at high Istim. This long-term SNP-induced increase in PS2 at high Istim was reduced by pretreatment with cycloheximide (5 mg kg-1, i.p.). These data, which provide the first demonstration that in situ manipulation of NO produces dual effects on neuronal responsiveness in a highly seizure-prone brain region, suggest that tonic levels of endogenous NO reduce the excitability of CA3 pyramidal neurones, whereas long-term overexposure to NO causes hyperexcitability possibly via genomic mechanisms.

ucb L059, a novel anticonvulsant, reduces bicuculline-induced hyperexcitability in rat hippocampal CA3 in vivo.

Local diffusion of bicuculline from a bicuculline-containing recording micropipette increased the orthodromic field population spike (PS2), elicited upon commissural stimulation in the hippocampal CA3 region of anaesthetized rats. This increase in PS2 was consistently reduced by 5.4 mg/kg of the novel anticonvulsant drug ucb L059 ((S)-alpha-ethyl-2-oxo-pyrrolidine acetamide), when injected i.v. 10 min prior to lowering in place the bicuculline-containing micropipette. ucb L059 had no effect on PS2 in the absence of bicuculline. Paired-pulse stimulation produced marked inhibition of the second PS2 at low interstimulus interval, an effect which was significantly reduced by bicuculline. Although ucb L059 reduced the effect of bicuculline on both PS2S elicited by paired stimulations, the drug did not alter the reduction by bicuculline of paired-pulse inhibition at low interstimulus interval. These results suggest that ucb L059 prevents increases in CA3 neuronal excitability by bicuculline through a non gamma-aminobutyric acid-ergic mechanism.

Input-output dependence reveals the components of GABAergic modulation in rat hippocampal CA3 in vivo.

Field population spikes, orthodromically elicited (PS2), upon commissural stimulation, in the hippocampal CA3 region of the anaesthetized rat, were determined at stimulation intensities (Istim) varying from 0.25 to 6 mA. A sharp, stimulation-dependent increase in the PS2 amplitude was observed with increasing Istim up to 0.5 mA, followed by an exponential decrease in PS2 at higher Istim, reaching a steady value with Istim above 3 mA. Intravenous (iv) infusion of bicuculline (BIC) increased the PS2, when elicited by Istim between 0.25 and 6 mA, though the stimulus-response (S-R) curve consistently presented a local minimum in between 0.5 to 1 mA. Baclofen (10 and 30 mg/kg iv) dose-dependently reduced PS2 at the lowest Istim (0.25 mA), both in control and in BIC-treated animals. At higher stimulation intensities, however, baclofen (30 mg/kg iv) increased PS2, while barely affecting the population spike in BIC-treated animals. The input-output dependence of this CA3 response appeared able to provide a convenient in vivo model revealing the components of GABAergic modulation.

Long-term adrenalectomy reduces hippocampal granule cell excitability in vivo.

Ten days after bilateral adrenalectomy (ADX), neural transmission between the perforant path and hippocampal dentate granule cells was severely impaired in the anaesthetized rat, in that the slope of the stimulus-response curve was reduced to less than half the value in sham controls, the stimulation current necessary to elicit a standard population spike (PS) field potential was increased approximately threefold, the amplitude of PS and its ratio to the slope of the field excitatory postsynaptic potential (EPSP) were reduced, and high-frequency tetanization (TET) of the perforant path resulted in potentiated PS with smaller amplitude and higher onset latency in ADX rats than in sham controls. However, the fractional increase of PS amplitude and its decay following TET were similar in 10 day-ADX and sham groups, from which it is inferred that long-term ADX entails a general decrease of dentate granule cell excitability, particularly at somatic membrane level, without specifically affecting the mechanism of long-term potentiation. None of the above changes occurred 24 h after ADX.

Dose dependence of glutaraldehyde-induced changes in the electrical properties of the amphibian skin.

The effects of the protein cross-linker glutaraldehyde (GA) on the transepithelial short-circuit current (Isc), conductance (Gt) and impedance of the isolated frog skin were investigated at GA concentrations between 0.1 and 10 mM, i.e. up to three orders of magnitude less than used in fixative procedures. Below 0.5 mM GA increases Isc, with large variations among preparata. Millimolar GA concentrations induce fairly reproducible irreversible inhibitions of Isc, which proceed for about 3 h. The rate of Isc decrease and the amplitudes of the initial drop and subsequent recovery depend on GA concentration in a sigmoidal dose-effect way, reaching saturation at 10 mM. At this GA concentration, Gt is increased up to 5 times the control value. Transepithelial impedance measurements confirm the decreases in epithelial resistance (Rm) and show significant increases in epithelial capacitance (Cm). Rm is diminished by 20% at 0.5 mM GA and by 75% at 10 mM GA, while Cm is maximally augmented by 55% at 2.5 mM GA. It is concluded that protein cross-linking by mild GA treatment is a convenient procedure for changing the electrical characteristics of epithelia.

Effects of millimolar concentrations of glutaraldehyde on the electrical properties of frog skin.

1. The effects of millimolar concentrations of glutaraldehyde on the electrophysiological properties of the epithelium of frog skin (Rana temporaria) were investigated. We recorded short-circuit current (Isc), transepithelial conductance (Gt) and impedance (Zt), fractional resistance (fRo) and the potential difference across the apical membrane (Vo). We used either Na+ or K+ as major mucosal cations to compare the effects on transepithelial Na+ and K+ currents (INa and IK) and thus on the apical Na+ and K+ permeabilities. 2. At concentrations above 0.005% (w/v) or 0.5 mM, glutaraldehyde irreversibly and completely inhibits both INa and IK within 2-3 h. The initial time courses of the inhibition of transepithelial currents following serosal and mucosal applications of the compound markedly differ. 3. Glutaraldehyde decreased Gt in sulphate Ringer solutions while it augmented Gt severalfold in chloride Ringer solution. 4. Measurements of the transepithelial impedance of tissues incubated with sulphate solutions showed that glutaraldehyde increased the resistances of both apical and basolateral membranes significantly. The capacitance of the apical membrane was augmented, while the basolateral membrane capacitance was drastically decreased. 5. Microelectrode impalements of the granulosum cells showed that glutaraldehyde decreased Vo by more than 40 mV and increased fRo, which reached values around 90%. 6. The role of free amino groups in ion-transporting proteins and the potential non-fixative uses of protein cross-linkers in epithelia are discussed.

Bioelectrical conversion in sensors with living cells.

Bacterial metabolism can drive various processes with biotechnological significance, like in the case of biosensors for probing organic compounds, or in biofuel cells. Here we present some of our results connected with the construction of a bacterial electrode with Pseudomonas sp. for probing glucose in body fluids, and those related to biofuel cells with anaerobic bacteria (Clostridium sp.). The output of the bioselective electrode linearly indicates glucose concentrations in the 2.5 x 10(-5)-1.25 x 10(-4) M domain, with a sensitivity limit of 10(-5) M. The time constant is 10 min, and it works for more than ten days. The amperometric response of biofuel cells with Clostridium sp. offers the electrical image of the state of the bacterial culture itself, thus being a promising tool for the automated control of the bacterial suspensions used in fermentative reactors.