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`Review: Is levetiracetam different from other
`antiepileptic drugs? Levetiracetam and its cellular
`mechanism of...
`
`Article in Therapeutic Advances in Neurological Disorders · July 2008
`
`DOI: 10.1177/1756285608094212 · Source: PubMed
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`

`
`Therapeutic Advances in Neurological Disorders
`
`Review
`
`Is levetiracetam different from other
`antiepileptic drugs? Levetiracetam and
`its cellular mechanism of action in
`epilepsy revisited
`
`Rainer Surges, Kirill E. Volynski and Matthew C. Walker
`
`Therapeutic Advances in
`Neurological Disorders
`
`(2008) 1(1) 13–24
`
`DOI: 10.1177/
`1756285608094212
`ß SAGE Publications 2008
`Los Angeles, London,
`New Delhi and Singapore
`
`Abstract: Levetiracetam (LEV) is a new antiepileptic drug that is clinically effective in
`generalized and partial epilepsy syndromes as sole or add-on medication. Nevertheless, its
`underlying mechanism of action is poorly understood. It has a unique preclinical profile;
`unlike other antiepileptic drugs (AEDs), it modulates seizure-activity in animal models of
`chronic epilepsy with no effect in most animal models of acute seizures. Yet it is effective
`in acute in-vitro ‘seizure’ models. A possible explanation for these dichotomous findings is
`that LEV has different mechanisms of actions, whether given acutely or chronically and in
`‘epileptic’ and control tissue. Here we review the general mechanism of action of AEDs,
`give an updated and critical overview about the experimental findings of LEV’s cellular
`targets (in particular the synaptic vesicular protein SV2A) and ask whether LEV represents a
`new class of AED.
`
`Keywords:
`ion channels
`
`levetiracetam, SV2A, antiepileptic drugs, synaptic transmission, epilepsy,
`
`Introduction
`Antiepileptic drug (AED) development has
`mainly taken place through trial and error.
`AEDs have been screened in animal models of
`seizures and epilepsy, often with an incomplete
`knowledge of their mechanism of action [Walker
`et al. 2004]. Indeed, the identification of drugs
`acting at putative ‘antiepileptic’
`targets has
`rarely translated into successful AED therapies,
`because the drugs are often poorly tolerated or
`have poor efficacy. Moreover, AEDs that were
`designed to act at specific targets (e.g., gaba-
`pentin, lamotrigine) work via different mechan-
`isms. Consequently, the underlying mechanism
`of action of an individual drug may only
`become apparent after its widespread clinical
`use. However, growing evidence suggests that
`many of the drugs that we use fall into one or
`more specific mechanistic groups – drugs that
`act at sodium channels, calcium channels or the
`GABAergic system [Walker and Fisher, 2004].
`Other putative and potential
`targets include
`potassium channels, hyperpolarization-activated
`cation channels, and glutamate receptors. Here
`we briefly review the mechanism of action of
`
`AEDs and ask whether levetiracetam (LEV)
`represents a new class of AED.
`
`Main targets for AED
`
`Sodium channels
`Sodium channels provide the major target for a
`number of AEDs including phenytoin, carbama-
`zepine, oxcarbazpine, and lamotrigine. Voltage-
`gated sodium channels are critical
`for action
`potential
`(AP) generation and propagation
`[Catterall, 2000a]. The sodium channel exists
`in three principal conformational states: at hyper-
`polarized potentials the channel is in the resting
`closed state; with depolarization the channel
`opens and permits the conduction of sodium
`ions; the channel then enters a nonconducting,
`inactivated state. This inactivation is removed
`(termed deinactivation) by hyperpolarization.
`In this manner, depolarization results in a tran-
`sient inward sodium current that rapidly inacti-
`vates. In addition to these three states, there is
`also a slow inactivated state, which occurs with
`sustained or repeated depolarizations. This state
`
`Correspondence to:
`Rainer Surges
`Department of Clinical and
`Experimental Epilepsy,
`Institute of Neurology,
`University College London,
`London WC1N 3BG, UK
`r.surges@ion.ucl.ac.uk
`
`Rainer Surges
`Kirill E. Volynski
`Matthew C. Walker
`Department of Clinical and
`Experimental Epilepsy,
`Institute of Neurology,
`University College of London
`
`http://tan.sagepub.com
`
`13
`
`Page 00002
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`

`
`Therapeutic Advances in Neurological Disorders
`
`is selectively enhanced by the new AED, lacosa-
`mide [Errington et al. 2008].
`
`Phenytoin, lamotrigine, oxcarbazepine, and car-
`bamazepine bind to and stabilize the inactivated
`state of
`the sodium channel
`[Kuo, 1998].
`This has two effects: a greater proportion of
`channels are inactive at hyperpolarized mem-
`brane potentials, and second there is a delay in
`deinactivation. The effect on the excitability of
`neurons is 2-fold. The rate at which an axon
`can ‘fire’ is critically determined by the rate at
`which the sodium channels deinactivate. If this
`time is increased, then the ‘refractory period’ is
`prolonged, inhibiting sustained repetitive firing
`[McLean and Macdonald, 1983]. In addition,
`since these drugs bind to channels in their inac-
`tive state, then the greater the number of chan-
`nels that have entered this state, the greater the
`drug binding. This results in a ‘use dependent’
`phenomenon in which repetitive firing results in
`greater amounts of the drug bound and so greater
`inhibition. In addition, these drugs inhibit the
`persistent
`sodium current, which mediates
`long-lasting depolarizations [Lampl et al. 1998].
`Other AEDs such as valproate, topiramate, and
`zonisamide may also have similar effects on
`sodium channels, but have been less well
`characterized.
`
`Calcium channels
`Calcium channels are also putative targets
`for AEDs, as they regulate not only neuronal
`excitability but also neurotransmitter
`release
`[Catterall, 2000b]. The voltage-gated calcium
`channels expressed in the brain can be subdivi-
`ded into four main classes, L-, P/Q-, N-, and
`T- type channels. L-, P/Q-, and N-type channels
`are high-voltage activated (HVA) channels that
`require significant depolarization to open, while
`the T-type channel
`is a low-voltage activated
`(LVA) channel and is opened by relatively small
`depolarizations.
`
`The L-type channels are mainly expressed post-
`synaptically. L-type channels are slowly inacti-
`vated thereby permitting sustained calcium
`entry following a depolarization. Calcium enter-
`ing through L-type calcium channels may play
`a role in activity-dependent gene expression and
`synaptic plasticity. Some AEDs (such as carba-
`mazepine) have been proposed to antagonise
`L-type calcium channels but the relevance of
`this
`to their antiepileptic effect
`is unclear
`[Ambrosio et al. 1999].
`
`N- and P/Q-type channels are expressed at syna-
`ptic boutons where they mediate calcium entry
`necessary for neurotransmitter release. These
`channels rapidly inactivate, resulting in brief
`calcium transients. This calcium entry then trig-
`gers exocytosis of presynaptic vesicles. N- and
`P/Q–type calcium channels can be modulated
`by G-protein linked receptors such as GABAB
`receptors. Inhibition of these channels would be
`expected to decrease neurotransmitter release.
`Gabapentin’s and pregabalin’s effect on HVA cal-
`cium channels is complex and novel; they both
`show strong and specific binding for the 2 aux-
`illary calcium channel subunit and may modulate
`P/Q-type calcium channels [Dooley et al. 2007].
`
`T-type calcium channels are activated at relatively
`hyperpolarized potentials. They open with small
`depolarization and then rapidly
`inactivate.
`They have been proposed to contribute to the
`generation of physiological rhythms within the
`thalamus, and have been implicated in the gen-
`eration of spike-wave discharges associated with
`absence epilepsy [McCormick and Contreras,
`2001]. There is evidence that ethosuximide med-
`iates its effect through binding to and stabilizing
`the inactivated state of the T-type calcium chan-
`nel [Gomora et al. 2001]. Other drugs such as
`zonisamide and valproate have also been sug-
`gested to act at this channel [Todorovic and
`Lingle, 1998; Suzuki et al. 1992].
`
`GABAergic system
`Gamma amino butyric acid (GABA) is the major
`inhibitory neurotransmitter in the brain. It is
`formed and degraded in the GABA shunt.
`Glutamic acid decarboxylase (GAD) converts
`glutamate to GABA. Promotion of GABA synth-
`esis has been proposed to contribute to the action
`of some AEDs including valproate [Lo¨ scher,
`1989].
`
`GABA is released into the synaptic space where
`it acts on two receptor types: ionotropic GABAA
`and metabotropic GABAB receptors (a third
`type, termed GABAC receptors, is present pre-
`dominantly in the retina) [Bormann, 2000].
`Benzodiazepines act at specific GABAA receptor
`subtypes [Mehta and Ticku, 1999], increasing
`the affinity of GABAA receptors for GABA, and
`the probability of receptor opening. Topiramate
`also potentiates GABAA receptor currents in a
`subunit specific manner [Simeone et al. 2006].
`Barbiturates
`are
`less
`selective
`for GABAA
`receptor
`subtypes,
`and
`prolong
`receptor
`
`14
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`
`Page 00003
`
`

`
`Review
`
`opening times. Drugs that act at GABAB recep-
`tors have been less useful as AEDs, probably
`because GABAB receptors have a complex func-
`tion acting postsynaptically to decrease neuronal
`excitability but also presynaptically decreasing
`GABA release.
`
`GABA is taken up by glial and neuronal GABA
`transporters, inhibition of which is another AED
`target (tiagabine) [Rekling et al. 1990]. Inside the
`cell, GABA is degraded by GABA transaminase
`to succinic semialdehyde, and inhibition of this
`enzyme by
`the AED vigabatrin increases
`GABAergic transmission [Gale and Iadarola,
`1980].
`
`Other targets
`
`Potassium channels
`Potassium channels form one of the most diverse
`groups of ion channels and have a critical role in
`determining neuronal excitability [ Jan and Jan,
`1997]. Persistent potassium currents play a cru-
`cial part in determining the resting membrane
`potential of neurons. Voltage-gated potassium
`channels can influence the resting membrane
`potential but also repolarize neurons following
`AP, thereby influencing neurotransmitter release.
`In addition, the rate of repolarization by potas-
`sium channels, affects the ability of a neuron to
`sustain rapid repetitive firing. Voltage-gated
`potassium channels in the brain can be subdivi-
`ded into: channels that rapidly activate and inac-
`tivate (A-type channels), channels that open
`upon depolarization but do not significantly inac-
`tivate (delayed rectifier channels) and channels
`that close upon depolarization but are open at
`the resting potential (inward rectifying channels).
`There are other potassium channels that are
`similar in structure to the voltage-gated potas-
`sium channel, but are opened by intracellular
`calcium (calcium-activated potassium channels
`that mediate the afterhyperpolarization) or by
`cyclic nucleotides (mainly present in the retina
`where they mediate photoreceptor responses).
`There are also specific potassium channels that
`are inactivated by acetylcholine – termed M-type
`channels. Although, modulation of potassium
`channels would seem to be an ideal target for
`AEDs, most drugs have no or poorly character-
`ized effects on potassium channels. However,
`phenytoin blocks the delayed rectifier potassium
`channels in neuroblastoma cells and retigabine,
`a putative AED, has as its main mode of action
`
`channels
`potentiation of potassium M-type
`[Wuttke et al. 2005; Tatulian et al. 2001; Nobile
`and Lagostena, 1998].
`
`HCN channels
`HCN channels are permeable to both potassium
`and sodium and mediate a current
`termed
`the H-current. These channels are activated
`at hyperpolarised potentials and deactivated at
`depolarized potentials. H-currents depolarize
`neurons from the resting membrane potential
`and have an important
`role in potentiating
`and maintaining oscillations
`[Robinson and
`Siegelbaum, 2003]. They may play a part in ter-
`minating thalamic oscillations and the generation
`of spike-wave discharges of absence epilepsy. The
`H-current is also highly expressed in dendrites
`where it shunts excitatory inputs. Lamotrigine
`has been shown to enhance the H-current in den-
`drites [Poolos et al. 2002]. Likewise, gabapentin
`has been demonstrated to increase the H-current
`in pyramidal neurons [Surges et al. 2003]. This
`may have two potentially antiepileptic effects: in
`the hippocampus it would inhibit excitatory
`transmission to the soma, explaining the efficacy
`of lamotrigine and gabapentin in partial epilepsy.
`In the thalamus,
`it may inhibit or terminate
`spike-wave discharges and,
`therefore, could
`explain the
`efficacy of
`lamotrigine against
`absence seizures.
`
`Glutamate and glutamate receptors
`Glutamate is the major excitatory transmitter in
`the central nervous system and acts at distinct
`receptor types: N-methyl-D-aspartate (NMDA),
`nonNMDA [consisting
`of
`alpha-
`amino-
`3-hydroxy-5-methyl-4-isoxazolepropionic
`acid
`(AMPA) and kainic acid (KA) sensitive recep-
`tors] and metabotropic glutamate receptors.
`Inhibition of these receptors would seem to be
`an ideal target for AEDs, but such compounds
`have been associated with unacceptable side-
`effects. NMDA receptors influence memory,
`cognition, and learning and NMDA receptor
`antagonists have had unacceptable side effects
`in clinical use. Felbamate and remacemide, how-
`ever, may modulate NMDA receptor-mediated
`transmission [Subramaniam et al. 1996; White
`et al. 1995].
`
`Topiramate at high concentrations acts at AMPA/
`kainate receptors; whether this is responsible for
`its antiepileptic effect or dose-related side effects
`is unknown [Angehagen et al. 2004]. Low doses
`of phenobarbitone have been shown to block
`
`http://tan.sagepub.com
`
`15
`
`Page 00004
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`

`
`Therapeutic Advances in Neurological Disorders
`
`AMPA receptors in the cerebral cortex [Sawada
`and Yamamoto, 1985], but the significance of
`this finding and its overall contribution towards
`the
`antiepileptic
`effects of phenobarbitone
`remains to be established. There are other
`drugs in clinical trials such as talampanel that
`are AMPA receptor antagonists.
`
`Levetiracetam
`LEV is a water soluble pyrrolidone derivative
`((S)--ethyl-2-oxo-pyrrolidine
`acetamide),
`whose chemical structure differs from other
`AEDs. Since its approval
`for clinical use in
`2002, LEV has become a widely used AED that
`is effective in partial and generalized epilepsy syn-
`dromes as sole or add-on medication [De Smedt
`et al. 2007]. Usual antiepileptic plasma concen-
`trations range from trough levels between 35 and
`100 mM (5.95–17 mg/ml) to peak levels between
`90 and 250 mM (15.3–42.5 mg/ml) [Rigo et al.
`2002; Patsalos, 2000].
`Importantly,
`serum
`levels of LEV are very similar to corresponding
`LEV levels found in the brain tissue of individual
`patients [Rambeck et al. 2006]. Unlike other
`AEDs, LEV is probably not a substrate for multi-
`drug transporters [Potschka et al. 2004].
`
`Except for rare instances of the treatment of
`acute seizures, AED therapy involves a regular
`daily, therefore chronic,
`intake of medication.
`Therefore, acute in-vitro and in-vivo experimen-
`tal paradigms do not necessarily reflect the clin-
`ical use of an AED. Moreover, there are various
`epilepsy-associated modifications of brain phy-
`siology, and therefore models of acute seizures
`differ
`from models
`of
`chronic
`epilepsy.
`Intriguingly, LEV modulates seizure activity in
`animal models of chronic epilepsy (kindling
`models, pilocarpine model, genetic absence epi-
`lepsy rats from Strasbourg GAERS) with no
`effect in most models of acute seizures [Glien
`et al. 2002; Klitgaard et al. 1998; Lo¨ scher and
`Ho¨ nack, 1993]. This is conisistent with the
`experimental observations that LEV only affects
`GABAA receptors from epileptic tissue or under
`conditions that occur during epilepsy, whereas it
`has no effect on GABAA receptors from controls
`[Palma et al. 2007; Rigo et al. 2002]. Taken
`together, these data suggest that LEV may pre-
`ferentially work with chronic application or under
`chronic epilepsy-associated conditions. However,
`most of the experiments to investigate LEV’s
`cellular mechanism of action have been per-
`formed by acute application, reporting its acute
`
`cellular effects. A further consideration is that
`LEV is now being proposed as an acute treatment
`for seizures. An intravenous formulation is avail-
`able [Ramael et al. 2006] that has already been
`shown to terminate status epilepticus after acute
`intravenous application [Knake et al. 2008].
`Interestingly, LEV is effective in one model of
`acute
`epilepsy
`(6 Hz
`psychomotor
`seizure
`model) [Shannon et al. 2005; Barton et al. 2001]
`with the maximal effect occurring 1 h after injec-
`tion [Barton et al. 2001]. These findings suggest
`that LEV can have a rapid-onset effect in some
`acute seizure models. One possible explanation
`for
`these dichotomous
`findings
`in animal
`models of acute and chronic epilepsy is that
`LEV may have different mechanisms of action
`whether given acutely or chronically and in epi-
`leptic and control tissue (see subsequently).
`
`Action of LEV on voltage-gated ion channels
`and regulation of intracellular ions
`Neuronal excitability and firing behavior are
`crucially shaped by voltage-gated ion channels.
`Acute application of LEV at a relatively low
`concentration (10 mM) did not alter neuronal
`properties such as membrane potential,
`input
`resistance, AP amplitude, AP duration, or fast
`and slow afterhyperpolarization of CA3 pyrami-
`dal cells [Birnstiel et al. 1997]. However, at
`higher concentrations (similar to those used in
`clinical practice) there is substantial experimental
`evidence that acutely applied LEV (and prolon-
`ged application for up to 1 h) modulates cellular
`targets that are important for neuronal excitabil-
`ity and synaptic transmission (cf. Tables 1 and 2).
`
`Voltage-gated ion channels
`HVA Ca2þ
`currents in different cell preparations
`(acutely isolated striatal, neocortical, and hippo-
`campal CA1 neurons, CA1 pyramidal neurons in
`slices) were inhibited by an average of 18–40%
`when LEV was acutely applied at different con-
`centrations (1–300 mM) [Costa et al. 2006; Pisani
`et al. 2004; Lukyanetz et al. 2002; Niespodziany
`et al. 2001]. Pharmacological separation of differ-
`ent HVA Ca2þ
`channel subtypes revealed that
`mainly N-type, and to a lesser extent P/Q-type
`calcium channels were affected [Costa et al.
`2006; Pisani et al. 2004; Lukyanetz et al. 2002].
`Changes in steady-state activation or inactivation
`properties were not observed [Lukyanetz et al.
`2002].
`In contrast, LEV did not modulate
`amplitudes,
`steady-state activation/inactivation
`properties or kinetics of T-type Ca2þ
`currents
`
`16
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`
`Page 00005
`
`

`
`Review
`
`Table 1. LEV effects on voltage-gated ion currents.
`
`Effect
`
`Concentration
`
`Application time Tissue
`
`Reference
`
`þ
`
`currents
`Na
`þ
`Fast Na
`currents
`
`Persistent
`þ
`Na
`currents
`Ca2þ
`HVA
`
`currents
`
`No effect
`
`No effect
`
`No effect
`
`No effect
`
`channel
`
`Inhibition by 30%
`Inhibition by 18% via
`N-type Ca2þ
`blockade
`Inhibition by 35%, mainly
`via blockade of N-type
`and to a lower extent
`of P/Q-type Ca2þ
`channels
`Inhibition by 40% (30%
`N-type, 10% P-type)
`
`LVA
`
`þ
`currents
`K
`Delayed rectifier
`þ
`current
`K
`A-type
`þ
`K
`current
`
`No effect on T-type
`Ca2þ
`currents
`Inhibition by 30%
`
`No effect
`
`10 mM to 1 mM
`
`100 mM
`
`1–500 mM
`
`10–100 mM
`
`32 mM
`
`200 mM
`
`100 mM
`
`Acute up to
`10 min
`Acute
`
`Probably acute
`
`Acute for
`up to 1 h
`
`Acute up to
`30 min
`Acute
`
`Acute
`
`Cultured neocortical
`neurons
`Acutely isolated CA1
`neurons
`Acutely isolated striatal
`neurons
`CA1 neurons in slices
`
`CA1 pyramidal neurons
`in slices
`Acutely isolated
`hippocampal CA1
`neurons
`Acutely isolated
`neocortical neurons
`
`Zona et al. 2001
`
`Madeja et al.
`2003
`Costa et al. 2006
`
`Niespodziany et al.
`2004
`
`Niespodziany et al.
`2001
`Lukyanetz et al.
`2002
`
`Pisani et al. 2004
`
`Half-maximal
`inhibition at
`22 mM
`32–100 mM
`
`Probably acute
`
`Acutely isolated striatal
`neurons
`
`Costa et al. 2006
`
`Acute for
`up to 40 min
`
`CA1 pyramidal neurons
`in slices
`
`Zona et al. 2001
`
`100 mM
`
`100 mM
`
`Acute
`
`Acute
`
`Acutely isolated CA1
`neurons
`Acutely isolated CA1
`neurons
`
`Madeja et al.
`2003
`Madeja et al.
`2003
`
`[Zona et al. 2001]. A reduction of N-type and
`P/Q-type calcium currents can lead to a decrease
`presynaptic Ca2þ
`-dependent
`processes
`in
`involved in neurotransmitter release. These chan-
`nels have been implicated in both ictogenesis and
`also epileptogenesis.
`
`þ
`
`currents were decreased by
`Delayed rectifier K
`about 30% when LEV was acutely applied to
`þ
`currents were
`CA1 neurons, whereas A-type K
`unaffected [Madeja et al. 2003]. The reduction of
`þ
`currents led to a reduction in
`delayed rectifier K
`repetitive AP generation and a slight prolonga-
`tion of AP duration. The relevance of this to its
`antiepileptic effect remains unknown, but may
`decrease neuronal firing.
`
`Unlike some classical AEDs, acute and prolon-
`ged application (for up to 1 h) of LEV at different
`concentrations (ranging from 1 mM–1 mM) in
`different
`cell preparations
`(acutely
`isolated
`striatal and hippocampal CA1 neurons, cultured
`neocortical neurons) had no effect on either
`
`þ
`
`stores and Cl
`
`currents [Costa et al.
`the amplitudes of fast Na
`2006; Madeja et al. 2003; Zona et al. 2001] or on
`steady-state activation and inactivation properties
`or current kinetics [Zona et al. 2001]. Likewise,
`þ
`currents were not affected
`persistent Na
`[Niespodziany et al. 2004]. However, activity-
`þ
`dependent inhibition (use-dependence) of Na
`currents has, to our knowledge, not been investi-
`gated, but given the above findings it is unlikely
`that LEV would have an effect.
`Intraneuronal Ca2þ
`exchanger
`Intriguingly, other cellular targets for LEV that
`are involved in the regulation of neuronal excit-
`ability have recently been identified, including
`intraneuronal calcium stores. These play an
`important role in the regulation of neuronal
`excitability, neurotransmission and synaptic plas-
`ticity as well as disease-related processes such as
`epileptogenesis and seizure-like activity [Bardo
`et al. 2006; Pal et al. 2001]. In most neurons
`Ca2þ
`-release
`from these
`stores
`is mainly
`
`
`
`
`/HCO3
`
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`
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`
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`Therapeutic Advances in Neurological Disorders
`
`Table 2. LEV effects on ligand-gated ion currents and other targets.
`
`Effect
`
`Concentration
`
`Application
`time
`
`Tissue
`
`Reference
`
`Carunchio et al.
`2007
`Rigo et al. 2002
`
`Palma et al. 2007
`
`Rigo et al. 2002
`
`Cultured cortical
`neurons
`
`Cultured hippocampal
`neurons
`Membrane preparations
`transplanted into frog
`oocytes
`Cultured hippocampal
`neurons
`
`Cultured hippocampal
`neurons
`PC12 cells (rat
`pheochromocytoma
`cells)
`CA3 neurons in slices
`
`Angehagen et al.
`2003
`Cataldi et al. 2005
`
`Leniger et al. 2004
`
`AMPA receptors
`
`Inhibition by 10–25%
`
`Complete reversal of
`zinc-induced inhibition
`Alleviation of run-down
`upon repetitive
`activation
`Complete reversal of
`zinc-induced inhibition
`
`Inhibition by 50%
`
`Inhibition by 25–50%
`
`200 mM
`
`30 mM
`
`0.5–100 mM
`
`Acute
`
`Acute
`
`Incubation
`for 3 h
`
`Half-maximal effect
`at 0.04 mM
`
`Acute
`
`32 mM
`
`10 mM
`
`After 5 min
`
`After 5 min
`
`Inhibition
`
`10–50 mM
`
`Up to 20 min
`
`GABAA receptors
`
`Glycine receptors
`Ca2þ
`stores
`Ryanodine-regulated
`Ca2þ
`release
`IP3-regulated
`Ca2þ
`release
`
`
`/HCO3
`exchanger
`
`Cl
`
`regulated by inositol (1,4,5)-triphosphate (IP3)
`and ryanodine receptors with varying relative
`contributions of each. Activation of ryanodine
`receptors by caffeine in mixed hippocampal cell
`cultures led to a transient increase of intracellular
`Ca2þ
`as well as to spontaneous bursts in hippo-
`slices. These Ca2þ
`transients were
`campal
`reduced by almost 50% after 5 min incubation
`with LEV and occurrence of spontaneous bursts
`in slices was delayed by LEV [Angehagen et al.
`2003].
`Furthermore, Gq-protein
`coupled
`IP3-dependent Ca2þ
`release in rat PC12 pheo-
`chromocytoma cells was inhibited by about
`25–50% after 5 min incubation with LEV
`[Cataldi et al. 2005]. Thus, inhibition of Ca2þ
`release from intraneuronal Ca2þ
`stores induced
`by LEV may contribute to its antiepileptic effects.
`
`The function of many proteins is pH dependent.
`Changes in intraneuronal pH modulate neuronal
`activity and intraneuronal acidification reduces
`in in-vitro preparations
`seizure-like
`activity
`[Bonnet et al. 2000]. In hippocampal slices
`acutely applied LEV for up to 20 min acidified
`the internal pH of CA3 neurons [Leniger et al.
`2004]. Moreover, LEV administration decreased
`the frequency of spontaneous AP and bursts
`induced by 4-aminopyridine. Both effects were
`reversible upon washout and the latter could be
`reversed by
`application of
`a membrane-
`permeable base. Further analysis revealed that
`the acidification in the presence of LEV was prob-
`þ
`-dependent
`ably linked to the blockade of the Na
`
`
`
`exchanger. This exchanger partici-
`/HCO3
`Cl
`pates in the extrusion of intracellular acid, thus
`a LEV-induced blockade was proposed to acidify
`the
`intracellular milieu,
`thereby
`reducing
`seizure-like activity [Leniger et al. 2004].
`
`Action of LEV on synaptic transmission
`AEDs can act at pre- or post-synaptic sites
`to modulate
`synaptic
`transmission. Evoked
`presynaptic release of neurotransmitters is trig-
`gered by an AP-induced calcium influx via
`P/Q-, N-,
`and R-type
`calcium channels.
`We have already described the effect of LEV on
`presynaptic calcium channels. In the following
`paragraphs we will
`further discuss LEV
`action on synaptic transmission including its
`interaction with presynaptic vesicular proteins
`and modulation of post-synaptic ligand-gated
`receptors.
`
`Synaptic vesicle binding site for LEV
`Lynch et al. (2004) showed that LEV specifically
`binds to the synaptic vesicle protein SV2A, wher-
`eas it does not bind to its two isoforms SV2B or
`SV2C. Moreover it was demonstrated that SV2A
`binding affinity of different LEV derivatives posi-
`tively correlated with their antiepileptic potency
`in different animal models of epilepsy [Kaminski
`et al. 2008; Lynch 2004]. These findings strongly
`suggest that LEV binding to SV2A is involved in
`its antiepileptic effect. Indeed, SV2A knock-out
`(KO) mice strains display a severe seizure
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`
`first postnatal week
`the
`after
`phenotype
`[Crowder et al. 1999; Janz et al. 1999] indicating
`that SV2A may normally regulate signaling cas-
`cades
`involved
`in
`seizure
`generation.
`Unfortunately no data on the actual CSF levels
`of different LEV derivatives were provided in the
`above correlation studies. Thus, it is still possible
`that the potency of different LEV derivatives in
`seizure-prevention was related to different CSF
`levels rather than to different binding affinities
`to SV2A. It is also unclear how these LEV deri-
`vatives act on voltage- or ligand-gated ion chan-
`nels involved in synaptic transmission.
`
`SV2 is a major vesicular protein that contains
`12 putative transmembrane regions and resem-
`bles membrane transporters. [Janz et al. 1999].
`Unfortunately to date, there are no available
`reports on the specific binding site for LEV on
`the SV2A molecule. Also the functions of SV2
`proteins
`in synaptic
`transmission are
`still
`debated. Electrophysiological recordings in cul-
`tured autaptic hippocampal neurons
`(which
`form synapses with themselves) from single and
`double SV2A/SV2B KO mice suggest several,
`somewhat disparate, possible mechanisms by
`which SV2 may regulate vesicular exocytosis.
`At high extracellular calcium concentrations
`(i.e., when the initial release probability is high)
`neurons from the SV2A/SV2B double KO exhib-
`ited no change in the initial probability of release
`but
`revealed a
`sustained increase
`in the
`AP-evoked synaptic transmission during trains
`of AP [Janz et al. 1999]. Importantly the increase
`could be partially reversed by loading presynaptic
`terminals with slow calcium buffer EGTA. These
`results prompted two alternative hypothesises:
`(i) the function of SV2s is to regulate presynaptic
`calcium levels during repetitive activity or
`(ii) SV2s function as targets for residual calcium
`in regulating vesicular exocytosis. In contrast,
`when synaptic transmission was assessed at phy-
`siological extracellular calcium concentrations,
`loss of SV2A, and SV2B led to reduced initial
`release probability but had no effect on steady-
`state responses during the trains of AP [Custer,
`2006]. These results suggest that SV2A may
`regulate priming of docked synaptic vesicles,
`a process that makes them ready for exocytosis,
`and thus selectively enhancing low frequency
`neurotransmission.
`
`Overall, although SV2A was identified as a spe-
`cific binding site for LEV, a direct demonstration
`of SV2A mediating the antiepileptic effect of
`
`LEV via a change in synaptic transmission is
`still lacking.
`
`Synaptic transmission in-vivo
`In-vivo studies are an important tool to judge the
`overall efficacy of AEDs in native environment of
`preserved neuronal circuitry. However, very often
`it is not possible to precisely control the AED
`concentrations during an in-vivo experiment.
`This in turn makes difficult dissecting specific
`AED actions on different signaling pathways.
`The effects of acute and chronic LEV application
`on neurochemical parameters
`in the mouse
`brain were examined after single or repeated
`intraperitoneal LEV injections [Sills et al. 1997].
`No effect of LEV on whole brain preparations was
`detected either on the overall GABA and gluta-
`mate concentrations or on the overall activities of
`GABA transaminase and GAD. In contrast, when
`studied in-vivo on a regional basis, acute LEV
`application had different effects within different
`brain regions with the most pronounced (and
`opposite) effects on GABA turnover
`in the
`et al. 1996].
`cortex and striatum [Lo¨ scher
`The implications for this finding are uncertain
`but suggest that LEV may operate through differ-
`ent mechanisms in different brain areas.
`
`Acute systemic application of LEV had no effect
`on evoked population spikes in the hippocampal
`CA3 region recorded in-vivo, but prevented the
`increase of
`the population-spikes induced by
`local application of bicuculline [Margineau and
`Wu¨ lfert, 1995]. Since the paired-pulse ratio in
`the presence or absence of bicuculline was not
`altered by LEV, LEV probably has little effect
`at presynaptic
`sites when applied acutely.
`Furthermore,
`acute LEV prevented
`the
`bicuculline-induced increase
`in population-
`spikes probably independent of a GABAergic
`pathway [Margineau and Wu¨ lfert, 1995]. In
`addition, in in-vivo experiments, acute systemic
`application of antiepileptic LEV doses also failed
`to alter evoked field potentials and GABAergic
`(GABAA and GABAB receptor) mechanisms in
`the hippocampal dentate gyrus [Margineau and
`Klitgaard, 2003].
`
`Synaptic transmission in-vitro
`As stated in the previous paragraph, there is no
`electrophysiological evidence for a direct effect
`of acutely applied LEV on the pre- or post-
`synaptic site of naı¨ve GABAergic synapses.
`Further, there is no direct LEV effect on naı¨ve
`GABAA receptor mediated inhibitory currents in
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`Therapeutic Advances in Neurological Disorders
`
`neuronal cell cultures, or in hippocampal and
`hypothalamic slices [Poulain and Margineanu,
`2002; Rigo et al. 2002; Birnstiel et al. 1997].
`Intriguingly, however, there have been two studies
`describing an effect of LEV on GABAA receptors
`that have undergone modifications associated
`with epilepsy. First, in hippocampi from epileptic
`brains GABAA receptor function can be impaired
`via allosteric inhibition by zinc, thereby reducing
`the overall inhibitory effect of GABA [Coulter
`2000]. This zinc-induced inhibition of GABAA
`receptors was fully reversed by acute application
`of LEV in cultured hippocampal neurons,
`whereas GABAA-induced currents in controls
`were unchanged [Rigo et al., 2002]. Second,
`Palma et al. (2007) prepared neuronal membranes
`from human tissue removed during brain surgery.
`They then micro-transplanted the membrane
`preparations into frog oocytes and investigated
`evoked GABAA receptor currents. The GABAA
`receptor currents
`from epileptic hippocampi
`were
`substantially
`impaired upon repeated
`GABA application without changes in decay
`time. This run-down was substantially reduced
`by pre-incubation with LEV for 3 h, suggesting
`stabilization of GABAA receptor currents during
`repeated activation. The stabilizing effect was also
`present with testing on GABAA receptor currents
`in neocortical pyramidal neurons in slices from
`temporal lobe epilepsy patients. T